Amplified fragment length polymorphism (AFLP) analysis was carried out in Thatcher, near isogenic lines (NILs) carrying different genes conferring resistance against wheat leaf rust, and TcLr45 × Thatcher F2 progenies were used to develop markers for Lr45 gene. Sixty AFLP primer combinations were screened and most of them provided clear amplification products, 31 primer combinations displayed polymorphism of TcLr45 in 23 NILs. Two AFLP markers closely linked to the gene Lr45 were acquired: P-AGG/M-GAG261 bp, which was found closely linked to the Lr45 locus at a distance of 0.6 cM on one side, and P-ACA/M-GGT105 bp, which was found at a distance of 1.3 cM on the other side. The specific bands were cloned and subsequently sequenced. The 261-bp fragment produced by P-AGG/M-GAG showed 86% similarity with the sequence of Vulgare Hort Ⅰ gene; the 105-bp fragment produced by P-ACA/M-GGT showed 96% similarity with the phosphatidylserine decarboxylase gene of the Triticum monococcum. Both included an open reading frame (ORF). Amplified fragment length polymorphism (AFLP) analysis was carried out in Thatcher, near isogenic lines (NILs) carrying different genes conferring resistance against wheat leaf rust, and TcLr45 × Thatcher F2 progenies were used to develop markers for Lr45 gene. Sixty AFLP primer combinations were Two clones were linked to the gene Lr45, respectively: P-AGG / M-GAG261 bp, which was found closely linked to the Lr45 locus at a distance of 0.6 cM on one side, and P-ACA / M-GGT105 bp, which was found at a distance of 1.3 cM on the other side. The specific bands were cloned and following sequenced. The 261-bp fragment produced by P-AGG / M-GAG showed 86% similarity with the sequence of Vulgare Hort I gene; the 105-bp fragment produced by P-ACA / M-GGT showed 96% similarity with the phosphatidylserine decarboxylase gene of the Triticum monococcum. Both in cluded an open reading frame (ORF).