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This research was aimed to develop AFLP markers co-segregated with gene Lr24 and validate the using for marker assisted selection (MAS).An F2 population developed from the cross between the resistant line TcLr24 and the susceptible line Thatcher was tested for resistance to the Puccinia triticina races BGQQ and SHRT using for genetic analysis and molecular marker.A total of 224 AFLP primer combinations were used to test the resistant and susceptible parents,as well as the resistant bulk and the susceptible bulk.Four AFLP markers,P-A GA/M-CTT289 bp,P-AGC/M-CA C188 bp,P-A GC/M- CAC162bp,and P-ACG/M-CGC239bp,were co-segregated with Lr24.The AFLP fragment from the primer combination P-ACG/M-CGC was cloned,sequenced and converted into a STS marker named as ASTS212.Thatcher backgrounded NILs and 115 varieties were examined by using this STS marker and the marker SCS1302607 developed by Gupta.5R615,5R616,1R13,and 1R17 were identified and validated to contain gene Lr24.The marker is dominant and may be useful in identification the resistance gene Lr24 in wheat and wheat breeding programs.