Wheat leaf rust is a prevalent foliar disease in wheat worldwide. Growing resistant cultivars is an effec-tive strategy to minimize the impact of leaf rust on yield and grain quality. Lr42 is a leaf rust resistance gene identified from Aegilops tauschii and is still effective against current predominant leaf rust races in the United States and many other countries. In this study, we developed diagnostic DNA markers for Lr42 using the sequence polymorphisms of a differentially expressed gene (TaRPM1) encoding a putative NB-ARC protein in the Lr42 candidate region identified by RNA-sequencing of two near-isogenic lines con-trasting in Lr42 alleles. Markers were designed based on a deletion mutation and a single nucleotide poly-morphism (SNP) in the gene. Haplotype analyses of the newly developed markers in the three diversity panels demonstrated that they are diagnostic for Lr42, and superior to previously used markers in selec-tion accuracy. These markers have the advantages of low cost and easy assay, and they are suitable for marker-assisted selection in breeding programs with either high- or low-throughput marker screening facilities.