Abstract: In order to study the feasibility of NBS profiling in leaf rust resistance gene, fifteen near-isogenic lines (NILs) and the susceptible parent Thatcher were selected as materials in the study. The NBS profiling system was established in wheat successfully and the polymorphism was analyzed. In the meanwhile, the TcLr35 wheat containing wheat leaf rust resistance gene Lr35 and Thatcher was used for the cDNA NBS profiling in order to uncover the RGA fragments related to Lr35 gene in the transcriptional level. The experimental results showed that the combination of MseⅠ/NBS2, MseⅠ/NBS5, MseⅠ/NBS7 could amplify the polymorphic bands and the rate of polymorphism was 26.4%, 26.4%, 23.5% respectively among the sixteen combinations of enzyme/primer; Fifty-two combinations of enzyme/primer were used to screen the specific fragment using cDNA NBS profiling between resistant and susceptible materials; several combinations could reflect the difference in TcLr35 and Thatcher. These results indicated that NBS profiling could be used for the study of wheat leaf rust resistance gene, which was the foundation to screen the wheat leaf rust resistance gene and the molecular markers..