<?xml version="1.0" encoding="us-ascii"?>
<!-- DFIN ActiveDisclosure(SM) XBRL Linkbase Document - http://www.dfinsolutions.com/ -->
<!-- Creation Date      : 2021-05-10T16:42:00.9025675+00:00 -->
<!-- Version            : 5.0.1.615 -->
<!-- Package ID         : 09990e539667404386f975bae234f735 -->
<!-- Copyright (c) 2021 Donnelley Financial Solutions, Inc. All Rights Reserved. -->
<link:linkbase xmlns:link="http://www.xbrl.org/2003/linkbase" xmlns:xbrldt="http://xbrl.org/2005/xbrldt" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance" xsi:schemaLocation="http://www.xbrl.org/2003/linkbase http://www.xbrl.org/2003/xbrl-linkbase-2003-12-31.xsd">
  <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#all" arcroleURI="http://xbrl.org/int/dim/arcrole/all" />
  <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-default" arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-default" />
  <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#dimension-domain" arcroleURI="http://xbrl.org/int/dim/arcrole/dimension-domain" />
  <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#domain-member" arcroleURI="http://xbrl.org/int/dim/arcrole/domain-member" />
  <link:arcroleRef xlink:type="simple" xlink:href="http://www.xbrl.org/2005/xbrldt-2005.xsd#hypercube-dimension" arcroleURI="http://xbrl.org/int/dim/arcrole/hypercube-dimension" />
  <link:roleRef xlink:type="simple" xlink:href="prld-20210331.xsd#TemplateLink" roleURI="http://preludetx.com/20210331/role/TemplateLink" />
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/StatementSTATEMENTOFBALANCESHEETSUNAUDITED" xlink:href="prld-20210331.xsd#Role_StatementSTATEMENTOFBALANCESHEETSUNAUDITED" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/StatementSTATEMENTOFBALANCESHEETSUNAUDITED">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LiabilitiesAndStockholdersEquity" xlink:type="locator" xlink:label="us-gaap_LiabilitiesAndStockholdersEquity" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockholdersEquity" xlink:type="locator" xlink:label="us-gaap_StockholdersEquity" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RetainedEarningsAccumulatedDeficit" xlink:type="locator" xlink:label="us-gaap_RetainedEarningsAccumulatedDeficit" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AdditionalPaidInCapitalCommonStock" xlink:type="locator" xlink:label="us-gaap_AdditionalPaidInCapitalCommonStock" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockValue" xlink:type="locator" xlink:label="us-gaap_CommonStockValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockholdersEquityAbstract" xlink:type="locator" xlink:label="us-gaap_StockholdersEquityAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommitmentsAndContingencies" xlink:type="locator" xlink:label="us-gaap_CommitmentsAndContingencies" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_Liabilities" xlink:type="locator" xlink:label="us-gaap_Liabilities" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_OperatingLeaseLiabilityNoncurrent" xlink:type="locator" xlink:label="us-gaap_OperatingLeaseLiabilityNoncurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_OtherLiabilitiesNoncurrent" xlink:type="locator" xlink:label="us-gaap_OtherLiabilitiesNoncurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LiabilitiesCurrent" xlink:type="locator" xlink:label="us-gaap_LiabilitiesCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_OperatingLeaseLiabilityCurrent" xlink:type="locator" xlink:label="us-gaap_OperatingLeaseLiabilityCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AccruedLiabilitiesCurrent" xlink:type="locator" xlink:label="us-gaap_AccruedLiabilitiesCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AccountsPayableCurrent" xlink:type="locator" xlink:label="us-gaap_AccountsPayableCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LiabilitiesCurrentAbstract" xlink:type="locator" xlink:label="us-gaap_LiabilitiesCurrentAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:type="locator" xlink:label="us-gaap_LiabilitiesAndStockholdersEquityAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_Assets" xlink:type="locator" xlink:label="us-gaap_Assets" />
    <link:loc xlink:href="prld-20210331.xsd#prld_DeferredOfferingCostsNonCurrent" xlink:type="locator" xlink:label="prld_DeferredOfferingCostsNonCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_OperatingLeaseRightOfUseAsset" xlink:type="locator" xlink:label="us-gaap_OperatingLeaseRightOfUseAsset" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PropertyPlantAndEquipmentNet" xlink:type="locator" xlink:label="us-gaap_PropertyPlantAndEquipmentNet" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AssetsCurrent" xlink:type="locator" xlink:label="us-gaap_AssetsCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PrepaidExpenseAndOtherAssetsCurrent" xlink:type="locator" xlink:label="us-gaap_PrepaidExpenseAndOtherAssetsCurrent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CashAndCashEquivalentsAtCarryingValue" xlink:type="locator" xlink:label="us-gaap_CashAndCashEquivalentsAtCarryingValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AssetsCurrentAbstract" xlink:type="locator" xlink:label="us-gaap_AssetsCurrentAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AssetsAbstract" xlink:type="locator" xlink:label="us-gaap_AssetsAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NonvotingCommonStockMember" xlink:type="locator" xlink:label="us-gaap_NonvotingCommonStockMember" />
    <link:loc xlink:href="prld-20210331.xsd#prld_VotingCommonStockMember" xlink:type="locator" xlink:label="prld_VotingCommonStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfStockDomain" xlink:type="locator" xlink:label="us-gaap_ClassOfStockDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfStockDomain" xlink:type="locator" xlink:label="us-gaap_ClassOfStockDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementClassOfStockAxis" xlink:type="locator" xlink:label="us-gaap_StatementClassOfStockAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementTable" xlink:type="locator" xlink:label="us-gaap_StatementTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementLineItems" xlink:type="locator" xlink:label="us-gaap_StatementLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" order="10200.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" order="10230.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain_2" order="10240.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" order="10260.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="prld_VotingCommonStockMember" order="10280.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_NonvotingCommonStockMember" order="10300.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_AssetsAbstract" order="10330.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_AssetsCurrentAbstract" order="10410.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_CashAndCashEquivalentsAtCarryingValue" order="10490.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_PrepaidExpenseAndOtherAssetsCurrent" order="10530.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsCurrentAbstract" xlink:to="us-gaap_AssetsCurrent" order="10570.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_PropertyPlantAndEquipmentNet" order="10610.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_OperatingLeaseRightOfUseAsset" order="10650.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="prld_DeferredOfferingCostsNonCurrent" order="10690.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AssetsAbstract" xlink:to="us-gaap_Assets" order="10730.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_LiabilitiesAndStockholdersEquityAbstract" order="10770.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="us-gaap_LiabilitiesCurrentAbstract" order="10850.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_AccountsPayableCurrent" order="10930.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_AccruedLiabilitiesCurrent" order="10970.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_OperatingLeaseLiabilityCurrent" order="11010.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesCurrentAbstract" xlink:to="us-gaap_LiabilitiesCurrent" order="11050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="us-gaap_OtherLiabilitiesNoncurrent" order="11090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="us-gaap_OperatingLeaseLiabilityNoncurrent" order="11130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="us-gaap_Liabilities" order="11170.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="us-gaap_CommitmentsAndContingencies" order="11210.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="us-gaap_StockholdersEquityAbstract" order="11250.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_CommonStockValue" order="11330.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_AdditionalPaidInCapitalCommonStock" order="11410.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_RetainedEarningsAccumulatedDeficit" order="11450.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StockholdersEquityAbstract" xlink:to="us-gaap_StockholdersEquity" order="11490.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LiabilitiesAndStockholdersEquityAbstract" xlink:to="us-gaap_LiabilitiesAndStockholdersEquity" order="11530.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/StatementSTATEMENTOFBALANCESHEETSPARENTHETICALUNAUDITED" xlink:href="prld-20210331.xsd#Role_StatementSTATEMENTOFBALANCESHEETSPARENTHETICALUNAUDITED" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/StatementSTATEMENTOFBALANCESHEETSPARENTHETICALUNAUDITED">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockSharesOutstanding" xlink:type="locator" xlink:label="us-gaap_CommonStockSharesOutstanding" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockSharesIssued" xlink:type="locator" xlink:label="us-gaap_CommonStockSharesIssued" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockSharesAuthorized" xlink:type="locator" xlink:label="us-gaap_CommonStockSharesAuthorized" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockParOrStatedValuePerShare" xlink:type="locator" xlink:label="us-gaap_CommonStockParOrStatedValuePerShare" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NonvotingCommonStockMember" xlink:type="locator" xlink:label="us-gaap_NonvotingCommonStockMember" />
    <link:loc xlink:href="prld-20210331.xsd#prld_VotingCommonStockMember" xlink:type="locator" xlink:label="prld_VotingCommonStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfStockDomain" xlink:type="locator" xlink:label="us-gaap_ClassOfStockDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementClassOfStockAxis" xlink:type="locator" xlink:label="us-gaap_StatementClassOfStockAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementTable" xlink:type="locator" xlink:label="us-gaap_StatementTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementLineItems" xlink:type="locator" xlink:label="us-gaap_StatementLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="prld_VotingCommonStockMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_NonvotingCommonStockMember" order="10170.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockParOrStatedValuePerShare" order="10260.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesAuthorized" order="10300.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesIssued" order="10340.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CommonStockSharesOutstanding" order="10380.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/StatementSTATEMENTSOFCHANGESINCONVERTIBLEPREFERREDSTOCKANDSTOCKHOLDERSEQUITYDEFICITUNAUDITED" xlink:href="prld-20210331.xsd#Role_StatementSTATEMENTSOFCHANGESINCONVERTIBLEPREFERREDSTOCKANDSTOCKHOLDERSEQUITYDEFICITUNAUDITED" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/StatementSTATEMENTSOFCHANGESINCONVERTIBLEPREFERREDSTOCKANDSTOCKHOLDERSEQUITYDEFICITUNAUDITED">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NetIncomeLoss" xlink:type="locator" xlink:label="us-gaap_NetIncomeLoss" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodSharesShareBasedCompensation" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodSharesShareBasedCompensation" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodValueShareBasedCompensation" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodValueShareBasedCompensation" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodValueNewIssues" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodValueNewIssues" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SharesOutstanding" xlink:type="locator" xlink:label="us-gaap_SharesOutstanding" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockholdersEquity" xlink:type="locator" xlink:label="us-gaap_StockholdersEquity" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_TemporaryEquitySharesOutstanding" xlink:type="locator" xlink:label="us-gaap_TemporaryEquitySharesOutstanding" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_TemporaryEquityCarryingAmountAttributableToParent" xlink:type="locator" xlink:label="us-gaap_TemporaryEquityCarryingAmountAttributableToParent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NonvotingCommonStockMember" xlink:type="locator" xlink:label="us-gaap_NonvotingCommonStockMember" />
    <link:loc xlink:href="prld-20210331.xsd#prld_VotingCommonStockMember" xlink:type="locator" xlink:label="prld_VotingCommonStockMember" />
    <link:loc xlink:href="prld-20210331.xsd#prld_SeriesBConvertiblePreferredStockMember" xlink:type="locator" xlink:label="prld_SeriesBConvertiblePreferredStockMember" />
    <link:loc xlink:href="prld-20210331.xsd#prld_SeriesAConvertiblePreferredStockMember" xlink:type="locator" xlink:label="prld_SeriesAConvertiblePreferredStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfStockDomain" xlink:type="locator" xlink:label="us-gaap_ClassOfStockDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementClassOfStockAxis" xlink:type="locator" xlink:label="us-gaap_StatementClassOfStockAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RetainedEarningsMember" xlink:type="locator" xlink:label="us-gaap_RetainedEarningsMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AdditionalPaidInCapitalMember" xlink:type="locator" xlink:label="us-gaap_AdditionalPaidInCapitalMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockMember" xlink:type="locator" xlink:label="us-gaap_CommonStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EquityComponentDomain" xlink:type="locator" xlink:label="us-gaap_EquityComponentDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EquityComponentDomain" xlink:type="locator" xlink:label="us-gaap_EquityComponentDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementEquityComponentsAxis" xlink:type="locator" xlink:label="us-gaap_StatementEquityComponentsAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementTable" xlink:type="locator" xlink:label="us-gaap_StatementTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementLineItems" xlink:type="locator" xlink:label="us-gaap_StatementLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementEquityComponentsAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_AdditionalPaidInCapitalMember" order="10130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_RetainedEarningsMember" order="10150.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" order="10160.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" order="10190.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="prld_SeriesAConvertiblePreferredStockMember" order="10210.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="prld_SeriesBConvertiblePreferredStockMember" order="10250.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="prld_VotingCommonStockMember" order="10290.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="us-gaap_NonvotingCommonStockMember" order="10330.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TemporaryEquityCarryingAmountAttributableToParent" order="10620.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_TemporaryEquitySharesOutstanding" order="10640.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockholdersEquity" order="10900.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SharesOutstanding" order="11000.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueStockOptionsExercised" order="11180.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesStockOptionsExercised" order="11320.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueNewIssues" order="11460.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" order="11600.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodValueShareBasedCompensation" order="11740.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesShareBasedCompensation" order="11880.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetIncomeLoss" order="12020.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/StatementSTATEMENTSOFCHANGESINCONVERTIBLEPREFERREDSTOCKANDSTOCKHOLDERSEQUITYDEFICITPARENTHETICALUNAUDITED" xlink:href="prld-20210331.xsd#Role_StatementSTATEMENTSOFCHANGESINCONVERTIBLEPREFERREDSTOCKANDSTOCKHOLDERSEQUITYDEFICITPARENTHETICALUNAUDITED" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/StatementSTATEMENTSOFCHANGESINCONVERTIBLEPREFERREDSTOCKANDSTOCKHOLDERSEQUITYDEFICITPARENTHETICALUNAUDITED">
    <link:loc xlink:href="prld-20210331.xsd#prld_PaymentsOfOfferingCosts" xlink:type="locator" xlink:label="prld_PaymentsOfOfferingCosts" />
    <link:loc xlink:href="prld-20210331.xsd#prld_SeriesBConvertiblePreferredStockMember" xlink:type="locator" xlink:label="prld_SeriesBConvertiblePreferredStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfStockDomain" xlink:type="locator" xlink:label="us-gaap_ClassOfStockDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementClassOfStockAxis" xlink:type="locator" xlink:label="us-gaap_StatementClassOfStockAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementTable" xlink:type="locator" xlink:label="us-gaap_StatementTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementLineItems" xlink:type="locator" xlink:label="us-gaap_StatementLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="prld_SeriesBConvertiblePreferredStockMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="prld_PaymentsOfOfferingCosts" order="10120.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/StatementSTATEMENTSOFCASHFLOWSUNAUDITED" xlink:href="prld-20210331.xsd#Role_StatementSTATEMENTSOFCASHFLOWSUNAUDITED" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/StatementSTATEMENTSOFCASHFLOWSUNAUDITED">
    <link:loc xlink:href="prld-20210331.xsd#prld_ReclassificationOfOfferingCostsPaidInPriorPeriod" xlink:type="locator" xlink:label="prld_ReclassificationOfOfferingCostsPaidInPriorPeriod" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CapitalExpendituresIncurredButNotYetPaid" xlink:type="locator" xlink:label="us-gaap_CapitalExpendituresIncurredButNotYetPaid" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SupplementalCashFlowInformationAbstract" xlink:type="locator" xlink:label="us-gaap_SupplementalCashFlowInformationAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" xlink:type="locator" xlink:label="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" xlink:type="locator" xlink:label="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NetCashProvidedByUsedInFinancingActivities" xlink:type="locator" xlink:label="us-gaap_NetCashProvidedByUsedInFinancingActivities" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ProceedsFromStockOptionsExercised" xlink:type="locator" xlink:label="us-gaap_ProceedsFromStockOptionsExercised" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RepaymentsOfLongTermCapitalLeaseObligations" xlink:type="locator" xlink:label="us-gaap_RepaymentsOfLongTermCapitalLeaseObligations" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ProceedsFromIssuanceOfConvertiblePreferredStock" xlink:type="locator" xlink:label="us-gaap_ProceedsFromIssuanceOfConvertiblePreferredStock" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:type="locator" xlink:label="us-gaap_ProceedsFromIssuanceOfCommonStock" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:type="locator" xlink:label="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NetCashProvidedByUsedInInvestingActivities" xlink:type="locator" xlink:label="us-gaap_NetCashProvidedByUsedInInvestingActivities" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" xlink:type="locator" xlink:label="us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:type="locator" xlink:label="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivities" xlink:type="locator" xlink:label="us-gaap_NetCashProvidedByUsedInOperatingActivities" />
    <link:loc xlink:href="prld-20210331.xsd#prld_IncreaseDecreaseInOperatingLeaseLiabilities" xlink:type="locator" xlink:label="prld_IncreaseDecreaseInOperatingLeaseLiabilities" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncreaseDecreaseInAccruedLiabilitiesAndOtherOperatingLiabilities" xlink:type="locator" xlink:label="us-gaap_IncreaseDecreaseInAccruedLiabilitiesAndOtherOperatingLiabilities" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncreaseDecreaseInAccountsPayable" xlink:type="locator" xlink:label="us-gaap_IncreaseDecreaseInAccountsPayable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncreaseDecreaseInPrepaidDeferredExpenseAndOtherAssets" xlink:type="locator" xlink:label="us-gaap_IncreaseDecreaseInPrepaidDeferredExpenseAndOtherAssets" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:type="locator" xlink:label="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensation" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensation" />
    <link:loc xlink:href="prld-20210331.xsd#prld_NoncashLeaseExpense" xlink:type="locator" xlink:label="prld_NoncashLeaseExpense" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DepreciationDepletionAndAmortization" xlink:type="locator" xlink:label="us-gaap_DepreciationDepletionAndAmortization" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="locator" xlink:label="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ProfitLoss" xlink:type="locator" xlink:label="us-gaap_ProfitLoss" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:type="locator" xlink:label="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" />
    <link:loc xlink:href="prld-20210331.xsd#prld_SeriesBConvertiblePreferredStockMember" xlink:type="locator" xlink:label="prld_SeriesBConvertiblePreferredStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfStockDomain" xlink:type="locator" xlink:label="us-gaap_ClassOfStockDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementClassOfStockAxis" xlink:type="locator" xlink:label="us-gaap_StatementClassOfStockAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementTable" xlink:type="locator" xlink:label="us-gaap_StatementTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementLineItems" xlink:type="locator" xlink:label="us-gaap_StatementLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_StatementTable" order="10170.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_StatementTable" xlink:to="us-gaap_StatementClassOfStockAxis" order="10200.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementClassOfStockAxis" xlink:to="us-gaap_ClassOfStockDomain" order="10230.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockDomain" xlink:to="prld_SeriesBConvertiblePreferredStockMember" order="10250.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" order="10280.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_ProfitLoss" order="10360.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" order="10400.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_DepreciationDepletionAndAmortization" order="10480.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="prld_NoncashLeaseExpense" order="10520.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AdjustmentsToReconcileNetIncomeLossToCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_ShareBasedCompensation" order="10560.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" order="10600.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInPrepaidDeferredExpenseAndOtherAssets" order="10680.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInAccountsPayable" order="10720.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="us-gaap_IncreaseDecreaseInAccruedLiabilitiesAndOtherOperatingLiabilities" order="10760.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncreaseDecreaseInOperatingCapitalAbstract" xlink:to="prld_IncreaseDecreaseInOperatingLeaseLiabilities" order="10800.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInOperatingActivitiesAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInOperatingActivities" order="10840.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" order="10880.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_PaymentsToAcquirePropertyPlantAndEquipment" order="10960.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInInvestingActivitiesAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInInvestingActivities" order="11000.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" order="11040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_ProceedsFromIssuanceOfCommonStock" order="11120.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_ProceedsFromIssuanceOfConvertiblePreferredStock" order="11160.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_RepaymentsOfLongTermCapitalLeaseObligations" order="11200.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_ProceedsFromStockOptionsExercised" order="11240.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_NetCashProvidedByUsedInFinancingActivitiesAbstract" xlink:to="us-gaap_NetCashProvidedByUsedInFinancingActivities" order="11280.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsPeriodIncreaseDecreaseIncludingExchangeRateEffect" order="11320.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_CashCashEquivalentsRestrictedCashAndRestrictedCashEquivalentsIncludingDisposalGroupAndDiscontinuedOperations" order="11360.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_StatementLineItems" xlink:to="us-gaap_SupplementalCashFlowInformationAbstract" order="11440.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SupplementalCashFlowInformationAbstract" xlink:to="us-gaap_CapitalExpendituresIncurredButNotYetPaid" order="11520.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SupplementalCashFlowInformationAbstract" xlink:to="prld_ReclassificationOfOfferingCostsPaidInPriorPeriod" order="11560.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/DisclosureSummaryOfSignificantAccountingPoliciesScheduleOfPotentiallyDilutiveSecuritiesExcludedFromComputationOfDilutedWeightedAverageSharesOfCommonStockOutstandingDetails" xlink:href="prld-20210331.xsd#Role_DisclosureSummaryOfSignificantAccountingPoliciesScheduleOfPotentiallyDilutiveSecuritiesExcludedFromComputationOfDilutedWeightedAverageSharesOfCommonStockOutstandingDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/DisclosureSummaryOfSignificantAccountingPoliciesScheduleOfPotentiallyDilutiveSecuritiesExcludedFromComputationOfDilutedWeightedAverageSharesOfCommonStockOutstandingDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" xlink:type="locator" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EmployeeStockOptionMember" xlink:type="locator" xlink:label="us-gaap_EmployeeStockOptionMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RestrictedStockMember" xlink:type="locator" xlink:label="us-gaap_RestrictedStockMember" />
    <link:loc xlink:href="prld-20210331.xsd#prld_SeriesBConvertiblePreferredStockMember" xlink:type="locator" xlink:label="prld_SeriesBConvertiblePreferredStockMember" />
    <link:loc xlink:href="prld-20210331.xsd#prld_SeriesAConvertiblePreferredStockMember" xlink:type="locator" xlink:label="prld_SeriesAConvertiblePreferredStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:type="locator" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AntidilutiveSecuritiesNameDomain" xlink:type="locator" xlink:label="us-gaap_AntidilutiveSecuritiesNameDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:type="locator" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:type="locator" xlink:label="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:to="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfAntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareTable" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareByAntidilutiveSecuritiesAxis" xlink:to="us-gaap_AntidilutiveSecuritiesNameDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="prld_SeriesAConvertiblePreferredStockMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="prld_SeriesBConvertiblePreferredStockMember" order="10110.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_RestrictedStockMember" order="10130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" order="10150.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareLineItems" xlink:to="us-gaap_AntidilutiveSecuritiesExcludedFromComputationOfEarningsPerShareAmount" order="10180.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/DisclosureSummaryOfSignificantAccountingPoliciesAdditionalInformationDetails" xlink:href="prld-20210331.xsd#Role_DisclosureSummaryOfSignificantAccountingPoliciesAdditionalInformationDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/DisclosureSummaryOfSignificantAccountingPoliciesAdditionalInformationDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShortTermLeaseCost" xlink:type="locator" xlink:label="us-gaap_ShortTermLeaseCost" />
    <link:loc xlink:href="prld-20210331.xsd#prld_OffsetAccruedExpenseAndOtherCurrentLiabilitiesToEliminateDeferredRent" xlink:type="locator" xlink:label="prld_OffsetAccruedExpenseAndOtherCurrentLiabilitiesToEliminateDeferredRent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_OperatingLeaseLiability" xlink:type="locator" xlink:label="us-gaap_OperatingLeaseLiability" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_OperatingLeaseRightOfUseAsset" xlink:type="locator" xlink:label="us-gaap_OperatingLeaseRightOfUseAsset" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ChangeInAccountingPrincipleAccountingStandardsUpdateImmaterialEffect" xlink:type="locator" xlink:label="us-gaap_ChangeInAccountingPrincipleAccountingStandardsUpdateImmaterialEffect" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ChangeInAccountingPrincipleAccountingStandardsUpdateAdoptionDate" xlink:type="locator" xlink:label="us-gaap_ChangeInAccountingPrincipleAccountingStandardsUpdateAdoptionDate" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ChangeInAccountingPrincipleAccountingStandardsUpdateAdopted" xlink:type="locator" xlink:label="us-gaap_ChangeInAccountingPrincipleAccountingStandardsUpdateAdopted" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AccountingStandardsUpdate201602Member" xlink:type="locator" xlink:label="us-gaap_AccountingStandardsUpdate201602Member" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_TypeOfAdoptionMember" xlink:type="locator" xlink:label="us-gaap_TypeOfAdoptionMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_TypeOfAdoptionMember" xlink:type="locator" xlink:label="us-gaap_TypeOfAdoptionMember_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" xlink:type="locator" xlink:label="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" />
    <link:loc xlink:href="prld-20210331.xsd#prld_SummaryOfSignificantAccountingPoliciesTable" xlink:type="locator" xlink:label="prld_SummaryOfSignificantAccountingPoliciesTable" />
    <link:loc xlink:href="prld-20210331.xsd#prld_SummaryOfSignificantAccountingPoliciesLineItems" xlink:type="locator" xlink:label="prld_SummaryOfSignificantAccountingPoliciesLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="prld_SummaryOfSignificantAccountingPoliciesLineItems" xlink:to="prld_SummaryOfSignificantAccountingPoliciesTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="prld_SummaryOfSignificantAccountingPoliciesTable" xlink:to="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" xlink:to="us-gaap_TypeOfAdoptionMember_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AdjustmentsForNewAccountingPronouncementsAxis" xlink:to="us-gaap_TypeOfAdoptionMember" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_TypeOfAdoptionMember" xlink:to="us-gaap_AccountingStandardsUpdate201602Member" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="prld_SummaryOfSignificantAccountingPoliciesLineItems" xlink:to="us-gaap_ChangeInAccountingPrincipleAccountingStandardsUpdateAdopted" order="10300.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="prld_SummaryOfSignificantAccountingPoliciesLineItems" xlink:to="us-gaap_ChangeInAccountingPrincipleAccountingStandardsUpdateAdoptionDate" order="10320.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="prld_SummaryOfSignificantAccountingPoliciesLineItems" xlink:to="us-gaap_ChangeInAccountingPrincipleAccountingStandardsUpdateImmaterialEffect" order="10340.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="prld_SummaryOfSignificantAccountingPoliciesLineItems" xlink:to="us-gaap_OperatingLeaseRightOfUseAsset" order="10390.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="prld_SummaryOfSignificantAccountingPoliciesLineItems" xlink:to="us-gaap_OperatingLeaseLiability" order="10410.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="prld_SummaryOfSignificantAccountingPoliciesLineItems" xlink:to="prld_OffsetAccruedExpenseAndOtherCurrentLiabilitiesToEliminateDeferredRent" order="10430.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="prld_SummaryOfSignificantAccountingPoliciesLineItems" xlink:to="us-gaap_ShortTermLeaseCost" order="10480.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/DisclosureFairValueOfFinancialInstrumentsScheduleOfAssetsAndLiabilitiesMeasuredAtFairValueOnRecurringBasisDetails" xlink:href="prld-20210331.xsd#Role_DisclosureFairValueOfFinancialInstrumentsScheduleOfAssetsAndLiabilitiesMeasuredAtFairValueOnRecurringBasisDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/DisclosureFairValueOfFinancialInstrumentsScheduleOfAssetsAndLiabilitiesMeasuredAtFairValueOnRecurringBasisDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisAssetValue" xlink:type="locator" xlink:label="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisAssetValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_MoneyMarketFundsMember" xlink:type="locator" xlink:label="us-gaap_MoneyMarketFundsMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:type="locator" xlink:label="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:type="locator" xlink:label="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CashAndCashEquivalentsAxis" xlink:type="locator" xlink:label="us-gaap_CashAndCashEquivalentsAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_FairValueInputsLevel3Member" xlink:type="locator" xlink:label="us-gaap_FairValueInputsLevel3Member" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_FairValueInputsLevel2Member" xlink:type="locator" xlink:label="us-gaap_FairValueInputsLevel2Member" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_FairValueInputsLevel1Member" xlink:type="locator" xlink:label="us-gaap_FairValueInputsLevel1Member" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="locator" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:type="locator" xlink:label="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:type="locator" xlink:label="us-gaap_FairValueByFairValueHierarchyLevelAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationTable" xlink:type="locator" xlink:label="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems" xlink:type="locator" xlink:label="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems" xlink:to="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationTable" xlink:to="us-gaap_FairValueByFairValueHierarchyLevelAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_FairValueByFairValueHierarchyLevelAxis" xlink:to="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel1Member" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel2Member" order="10110.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueMeasurementsFairValueHierarchyDomain" xlink:to="us-gaap_FairValueInputsLevel3Member" order="10130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationTable" xlink:to="us-gaap_CashAndCashEquivalentsAxis" order="10140.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_CashAndCashEquivalentsAxis" xlink:to="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember_2" order="10150.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_CashAndCashEquivalentsAxis" xlink:to="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" order="10170.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_RestrictedCashAndCashEquivalentsCashAndCashEquivalentsMember" xlink:to="us-gaap_MoneyMarketFundsMember" order="10190.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_FairValueAssetsMeasuredOnRecurringBasisUnobservableInputReconciliationLineItems" xlink:to="us-gaap_FairValueMeasurementWithUnobservableInputsReconciliationRecurringBasisAssetValue" order="10240.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/DisclosureCommonStockAdditionalInformationDetails" xlink:href="prld-20210331.xsd#Role_DisclosureCommonStockAdditionalInformationDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/DisclosureCommonStockAdditionalInformationDetails">
    <link:loc xlink:href="prld-20210331.xsd#prld_BeneficialOwnershipPercentage" xlink:type="locator" xlink:label="prld_BeneficialOwnershipPercentage" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockVotingRights" xlink:type="locator" xlink:label="us-gaap_CommonStockVotingRights" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ProceedsFromIssuanceOfCommonStock" xlink:type="locator" xlink:label="us-gaap_ProceedsFromIssuanceOfCommonStock" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SaleOfStockPricePerShare" xlink:type="locator" xlink:label="us-gaap_SaleOfStockPricePerShare" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StockIssuedDuringPeriodSharesNewIssues" xlink:type="locator" xlink:label="us-gaap_StockIssuedDuringPeriodSharesNewIssues" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_MaximumMember" xlink:type="locator" xlink:label="srt_MaximumMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_MinimumMember" xlink:type="locator" xlink:label="srt_MinimumMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_RangeMember" xlink:type="locator" xlink:label="srt_RangeMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_RangeMember" xlink:type="locator" xlink:label="srt_RangeMember_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_RangeAxis" xlink:type="locator" xlink:label="srt_RangeAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_CommonStockMember" xlink:type="locator" xlink:label="us-gaap_CommonStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EquityComponentDomain" xlink:type="locator" xlink:label="us-gaap_EquityComponentDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_StatementEquityComponentsAxis" xlink:type="locator" xlink:label="us-gaap_StatementEquityComponentsAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IPOMember" xlink:type="locator" xlink:label="us-gaap_IPOMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SaleOfStockNameOfTransactionDomain" xlink:type="locator" xlink:label="us-gaap_SaleOfStockNameOfTransactionDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SubsidiarySaleOfStockAxis" xlink:type="locator" xlink:label="us-gaap_SubsidiarySaleOfStockAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfStockByClassTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfStockByClassTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ClassOfStockLineItems" xlink:type="locator" xlink:label="us-gaap_ClassOfStockLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ScheduleOfStockByClassTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_SubsidiarySaleOfStockAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_SubsidiarySaleOfStockAxis" xlink:to="us-gaap_SaleOfStockNameOfTransactionDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_SaleOfStockNameOfTransactionDomain" xlink:to="us-gaap_IPOMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="us-gaap_StatementEquityComponentsAxis" order="10100.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_StatementEquityComponentsAxis" xlink:to="us-gaap_EquityComponentDomain" order="10130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_EquityComponentDomain" xlink:to="us-gaap_CommonStockMember" order="10150.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfStockByClassTable" xlink:to="srt_RangeAxis" order="10220.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember_2" order="10230.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" order="10250.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" order="10270.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" order="10290.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_StockIssuedDuringPeriodSharesNewIssues" order="10320.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_SaleOfStockPricePerShare" order="10340.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_ProceedsFromIssuanceOfCommonStock" order="10360.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="us-gaap_CommonStockVotingRights" order="10410.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ClassOfStockLineItems" xlink:to="prld_BeneficialOwnershipPercentage" order="10430.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/DisclosureCommitmentsAdditionalInformationDetails" xlink:href="prld-20210331.xsd#Role_DisclosureCommitmentsAdditionalInformationDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/DisclosureCommitmentsAdditionalInformationDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DefinedContributionPlanEmployerDiscretionaryContributionAmount" xlink:type="locator" xlink:label="us-gaap_DefinedContributionPlanEmployerDiscretionaryContributionAmount" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DefinedContributionPlanEmployerMatchingContributionPercent" xlink:type="locator" xlink:label="us-gaap_DefinedContributionPlanEmployerMatchingContributionPercent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_DefinedContributionPlanMaximumAnnualContributionsPerEmployeePercent" xlink:type="locator" xlink:label="us-gaap_DefinedContributionPlanMaximumAnnualContributionsPerEmployeePercent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_OperatingLeaseExpense" xlink:type="locator" xlink:label="us-gaap_OperatingLeaseExpense" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LesseeOperatingLeaseOptionToExtend" xlink:type="locator" xlink:label="us-gaap_LesseeOperatingLeaseOptionToExtend" />
    <link:loc xlink:href="prld-20210331.xsd#prld_OperatingLeaseEstimatedIncrementalBorrowingRatePercent" xlink:type="locator" xlink:label="prld_OperatingLeaseEstimatedIncrementalBorrowingRatePercent" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LesseeOperatingLeaseRenewalTerm" xlink:type="locator" xlink:label="us-gaap_LesseeOperatingLeaseRenewalTerm" />
    <link:loc xlink:href="prld-20210331.xsd#prld_OperatingLeaseExpiringDecember2022Member" xlink:type="locator" xlink:label="prld_OperatingLeaseExpiringDecember2022Member" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LeaseContractualTermDomain" xlink:type="locator" xlink:label="us-gaap_LeaseContractualTermDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LeaseContractualTermDomain" xlink:type="locator" xlink:label="us-gaap_LeaseContractualTermDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_LeaseContractualTermAxis" xlink:type="locator" xlink:label="us-gaap_LeaseContractualTermAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_OtherCommitmentsTable" xlink:type="locator" xlink:label="us-gaap_OtherCommitmentsTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_OtherCommitmentsLineItems" xlink:type="locator" xlink:label="us-gaap_OtherCommitmentsLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_OtherCommitmentsLineItems" xlink:to="us-gaap_OtherCommitmentsTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_OtherCommitmentsTable" xlink:to="us-gaap_LeaseContractualTermAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_LeaseContractualTermAxis" xlink:to="us-gaap_LeaseContractualTermDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_LeaseContractualTermAxis" xlink:to="us-gaap_LeaseContractualTermDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_LeaseContractualTermDomain" xlink:to="prld_OperatingLeaseExpiringDecember2022Member" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OtherCommitmentsLineItems" xlink:to="us-gaap_LesseeOperatingLeaseRenewalTerm" order="10120.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OtherCommitmentsLineItems" xlink:to="prld_OperatingLeaseEstimatedIncrementalBorrowingRatePercent" order="10140.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OtherCommitmentsLineItems" xlink:to="us-gaap_LesseeOperatingLeaseOptionToExtend" order="10160.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OtherCommitmentsLineItems" xlink:to="us-gaap_OperatingLeaseExpense" order="10210.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OtherCommitmentsLineItems" xlink:to="us-gaap_DefinedContributionPlanMaximumAnnualContributionsPerEmployeePercent" order="10280.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OtherCommitmentsLineItems" xlink:to="us-gaap_DefinedContributionPlanEmployerMatchingContributionPercent" order="10300.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_OtherCommitmentsLineItems" xlink:to="us-gaap_DefinedContributionPlanEmployerDiscretionaryContributionAmount" order="10320.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/DisclosureStockBasedCompensationAdditionalInformationDetails" xlink:href="prld-20210331.xsd#Role_DisclosureStockBasedCompensationAdditionalInformationDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/DisclosureStockBasedCompensationAdditionalInformationDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions" xlink:type="locator" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" xlink:type="locator" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions" xlink:type="locator" xlink:label="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="locator" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1" xlink:type="locator" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardSharesIssuedInPeriod" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardSharesIssuedInPeriod" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage" xlink:type="locator" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" />
    <link:loc xlink:href="prld-20210331.xsd#prld_PercentageOfSharesOutstandingUsedToCalculateAnnualIncreaseInNumberOfSharesThatCanBeIssued" xlink:type="locator" xlink:label="prld_PercentageOfSharesOutstandingUsedToCalculateAnnualIncreaseInNumberOfSharesThatCanBeIssued" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" xlink:type="locator" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardTermsOfAward" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardTermsOfAward" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" />
    <link:loc xlink:href="prld-20210331.xsd#prld_NumberOfEquityIncentivePlans" xlink:type="locator" xlink:label="prld_NumberOfEquityIncentivePlans" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RestrictedStockMember" xlink:type="locator" xlink:label="us-gaap_RestrictedStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EmployeeStockOptionMember" xlink:type="locator" xlink:label="us-gaap_EmployeeStockOptionMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AwardTypeAxis" xlink:type="locator" xlink:label="us-gaap_AwardTypeAxis" />
    <link:loc xlink:href="prld-20210331.xsd#prld_TwoThousandTwentyEmployeeStockPurchasePlanMember" xlink:type="locator" xlink:label="prld_TwoThousandTwentyEmployeeStockPurchasePlanMember" />
    <link:loc xlink:href="prld-20210331.xsd#prld_TwoThousandTwentyEquityIncentivePlanMember" xlink:type="locator" xlink:label="prld_TwoThousandTwentyEquityIncentivePlanMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PlanNameDomain" xlink:type="locator" xlink:label="us-gaap_PlanNameDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PlanNameDomain" xlink:type="locator" xlink:label="us-gaap_PlanNameDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_PlanNameAxis" xlink:type="locator" xlink:label="us-gaap_PlanNameAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_MinimumMember" xlink:type="locator" xlink:label="srt_MinimumMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_MaximumMember" xlink:type="locator" xlink:label="srt_MaximumMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_RangeMember" xlink:type="locator" xlink:label="srt_RangeMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/srt/2020/elts/srt-2020-01-31.xsd#srt_RangeAxis" xlink:type="locator" xlink:label="srt_RangeAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationAwardTrancheOneMember" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationAwardTrancheOneMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_VestingDomain" xlink:type="locator" xlink:label="us-gaap_VestingDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_VestingDomain" xlink:type="locator" xlink:label="us-gaap_VestingDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_VestingAxis" xlink:type="locator" xlink:label="us-gaap_VestingAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_VestingAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_VestingAxis" xlink:to="us-gaap_VestingDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_VestingDomain" xlink:to="us-gaap_ShareBasedCompensationAwardTrancheOneMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="srt_RangeAxis" order="10100.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="srt_RangeAxis" xlink:to="srt_RangeMember" order="10130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MaximumMember" order="10150.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="srt_RangeMember" xlink:to="srt_MinimumMember" order="10170.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_PlanNameAxis" order="10180.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain_2" order="10190.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_PlanNameAxis" xlink:to="us-gaap_PlanNameDomain" order="10210.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="prld_TwoThousandTwentyEquityIncentivePlanMember" order="10230.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_PlanNameDomain" xlink:to="prld_TwoThousandTwentyEmployeeStockPurchasePlanMember" order="10390.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" order="10420.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain_2" order="10430.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" order="10450.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" order="10470.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockMember" order="10610.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="prld_NumberOfEquityIncentivePlans" order="10820.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardNumberOfSharesAvailableForGrant" order="10840.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardTermsOfAward" order="10860.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardExpirationPeriod" order="10880.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="prld_PercentageOfSharesOutstandingUsedToCalculateAnnualIncreaseInNumberOfSharesThatCanBeIssued" order="10900.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardAwardVestingPeriod1" order="10920.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardAwardVestingRightsPercentage" order="10960.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardSharesIssuedInPeriod" order="11020.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsOutstandingIntrinsicValue" order="11070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardOptionsExercisableIntrinsicValue1" order="11090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsGrantsInPeriodWeightedAverageGrantDateFairValue" order="11140.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardOptionsExercisesInPeriodTotalIntrinsicValue" order="11180.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" order="11220.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedStockOptions" order="11260.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedPeriodForRecognition1" order="11300.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_EmployeeServiceShareBasedCompensationNonvestedAwardsTotalCompensationCostNotYetRecognizedShareBasedAwardsOtherThanOptions" order="11480.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/DisclosureStockBasedCompensationScheduleOfStockBasedCompensationExpenseRecordedInStatementsOfOperationsDetails" xlink:href="prld-20210331.xsd#Role_DisclosureStockBasedCompensationScheduleOfStockBasedCompensationExpenseRecordedInStatementsOfOperationsDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/DisclosureStockBasedCompensationScheduleOfStockBasedCompensationExpenseRecordedInStatementsOfOperationsDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AllocatedShareBasedCompensationExpense" xlink:type="locator" xlink:label="us-gaap_AllocatedShareBasedCompensationExpense" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_GeneralAndAdministrativeExpenseMember" xlink:type="locator" xlink:label="us-gaap_GeneralAndAdministrativeExpenseMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ResearchAndDevelopmentExpenseMember" xlink:type="locator" xlink:label="us-gaap_ResearchAndDevelopmentExpenseMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncomeStatementLocationDomain" xlink:type="locator" xlink:label="us-gaap_IncomeStatementLocationDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncomeStatementLocationDomain" xlink:type="locator" xlink:label="us-gaap_IncomeStatementLocationDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_IncomeStatementLocationAxis" xlink:type="locator" xlink:label="us-gaap_IncomeStatementLocationAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_IncomeStatementLocationAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_IncomeStatementLocationAxis" xlink:to="us-gaap_IncomeStatementLocationDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_ResearchAndDevelopmentExpenseMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_IncomeStatementLocationDomain" xlink:to="us-gaap_GeneralAndAdministrativeExpenseMember" order="10110.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_AllocatedShareBasedCompensationExpense" order="10140.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/DisclosureStockBasedCompensationSummaryOfInformationAboutStockOptionsOutstandingDetails" xlink:href="prld-20210331.xsd#Role_DisclosureStockBasedCompensationSummaryOfInformationAboutStockOptionsOutstandingDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/DisclosureStockBasedCompensationSummaryOfInformationAboutStockOptionsOutstandingDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeExercisableOptionsWeightedAverageExercisePrice1" xlink:type="locator" xlink:label="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeExercisableOptionsWeightedAverageExercisePrice1" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfExercisableOptions" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfExercisableOptions" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageExercisePriceBeginningBalance1" xlink:type="locator" xlink:label="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageExercisePriceBeginningBalance1" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageRemainingContractualTerm2" xlink:type="locator" xlink:label="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageRemainingContractualTerm2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfOutstandingOptions" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfOutstandingOptions" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeUpperRangeLimit" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeUpperRangeLimit" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLowerRangeLimit" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLowerRangeLimit" />
    <link:loc xlink:href="prld-20210331.xsd#prld_ExercisePriceRangeThreeMember" xlink:type="locator" xlink:label="prld_ExercisePriceRangeThreeMember" />
    <link:loc xlink:href="prld-20210331.xsd#prld_ExercisePriceRangeTwoMember" xlink:type="locator" xlink:label="prld_ExercisePriceRangeTwoMember" />
    <link:loc xlink:href="prld-20210331.xsd#prld_ExercisePriceRangeOneMember" xlink:type="locator" xlink:label="prld_ExercisePriceRangeOneMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain_2" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-default" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain_2" order="10050.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansByExercisePriceRangeAxis" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:to="prld_ExercisePriceRangeOneMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:to="prld_ExercisePriceRangeTwoMember" order="10110.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeDomain" xlink:to="prld_ExercisePriceRangeThreeMember" order="10130.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeLowerRangeLimit" order="10160.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeUpperRangeLimit" order="10280.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfOutstandingOptions" order="10400.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageRemainingContractualTerm2" order="10520.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeOutstandingOptionsWeightedAverageExercisePriceBeginningBalance1" order="10640.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeNumberOfExercisableOptions" order="10760.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationSharesAuthorizedUnderStockOptionPlansExercisePriceRangeExercisableOptionsWeightedAverageExercisePrice1" order="10880.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/DisclosureStockBasedCompensationScheduleOfFairValueOfEachOptionEstimatedOnDateOfGrantUsingWeightedAverageAssumptionsDetails" xlink:href="prld-20210331.xsd#Role_DisclosureStockBasedCompensationScheduleOfFairValueOfEachOptionEstimatedOnDateOfGrantUsingWeightedAverageAssumptionsDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/DisclosureStockBasedCompensationScheduleOfFairValueOfEachOptionEstimatedOnDateOfGrantUsingWeightedAverageAssumptionsDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" xlink:type="locator" xlink:label="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_EmployeeStockOptionMember" xlink:type="locator" xlink:label="us-gaap_EmployeeStockOptionMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AwardTypeAxis" xlink:type="locator" xlink:label="us-gaap_AwardTypeAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_EmployeeStockOptionMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedVolatilityRate" order="10180.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsRiskFreeInterestRate" order="10220.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_SharebasedCompensationArrangementBySharebasedPaymentAwardFairValueAssumptionsExpectedTerm1" order="10260.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardFairValueAssumptionsExpectedDividendRate" order="10300.00" priority="2" use="optional" />
  </link:definitionLink>
  <link:roleRef roleURI="http://preludetx.com/20210331/taxonomy/role/DisclosureStockBasedCompensationSummaryOfActivityRelatedToRSAStockBasedPaymentAwardsDetails" xlink:href="prld-20210331.xsd#Role_DisclosureStockBasedCompensationSummaryOfActivityRelatedToRSAStockBasedPaymentAwardsDetails" xlink:type="simple" />
  <link:definitionLink xlink:type="extended" xlink:role="http://preludetx.com/20210331/taxonomy/role/DisclosureStockBasedCompensationSummaryOfActivityRelatedToRSAStockBasedPaymentAwardsDetails">
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_RestrictedStockMember" xlink:type="locator" xlink:label="us-gaap_RestrictedStockMember" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_AwardTypeAxis" xlink:type="locator" xlink:label="us-gaap_AwardTypeAxis" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:type="locator" xlink:label="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" />
    <link:loc xlink:href="http://xbrl.fasb.org/us-gaap/2020/elts/us-gaap-2020-01-31.xsd#us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:type="locator" xlink:label="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/all" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" order="10010.00" priority="2" use="optional" xbrldt:contextElement="segment" xbrldt:closed="true" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/hypercube-dimension" xlink:from="us-gaap_ScheduleOfShareBasedCompensationArrangementsByShareBasedPaymentAwardTable" xlink:to="us-gaap_AwardTypeAxis" order="10040.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/dimension-domain" xlink:from="us-gaap_AwardTypeAxis" xlink:to="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" order="10070.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementsByShareBasedPaymentAwardAwardTypeAndPlanNameDomain" xlink:to="us-gaap_RestrictedStockMember" order="10090.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedNumber" order="10260.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriod" order="10300.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsNonvestedWeightedAverageGrantDateFairValue" order="10410.00" priority="2" use="optional" />
    <link:definitionArc xlink:type="arc" xlink:arcrole="http://xbrl.org/int/dim/arcrole/domain-member" xlink:from="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardLineItems" xlink:to="us-gaap_ShareBasedCompensationArrangementByShareBasedPaymentAwardEquityInstrumentsOtherThanOptionsVestedInPeriodWeightedAverageGrantDateFairValue" order="10430.00" priority="2" use="optional" />
  </link:definitionLink>
</link:linkbase>
