Wheat streak mosaic virus (WSMV) can cause significant yield loss in wheat (Triticum aestivum L.) in the Great Plains of North America. A recently identified WSMV resistance gene, Wsm2, was mapped to chromosome 3BS in germplasm line 'CO960293–2'. Effective genetic markers tightly linked to the gene will enhance the selection of WSMV-resistant lines through marker-assisted selection. We have mapped Wsm2 using a high-density map developed from the wheat 90K Infinium iSelect single-nucleotide polymorphism (SNP) array with recombinant inbred lines from the cross between CO960293–2 and susceptible cultivar 'TAM 111'. Array-based SNPs that mapped within 4 cM of Wsm2 on chromosome 3BS were converted to Kompetitive Allele Specific Polymerase Chain Reaction (KASP) assays in this study. Six KASP SNPs were validated in two doubled haploid populations developed from crosses of 'RonL' × 'Ripper' and 'Snowmass' × 'Antero'. RonL and Snowmass possess the Wsm2 gene from CO960293–2. Three closely linked KASP SNPs, converted from IAAV6442, BS00018764_51, and wsnp_Ra_c16264_24873670, showed high sensitivity and specificity (0.83 ≤ sensitivity ≤ 0.97, 0.89 ≤ specificity ≤ 0.99). The latter two were also validated in six F2 breeding populations. These three KASP SNPs were effective in differentiating resistant and susceptible genotypes. Comparative mapping was performed using sequences of SNPs flanking Wsm2 and identified candidate genes and regions in Brachypodium and rice (Oryza sativa L. ssp. japonica). The KASP SNPs developed in this study should be useful for marker-assisted selection of Wsm2 in wheat breeding programs, and the newly constructed map will also facilitate map based cloning of Wsm2. Resources in this dataset:Resource Title: Supplemental Table S1: A list of candidate genes identified in the region between wheat markers GENE-1856_1005 and Tdurum_contig57914_1144 2 to Brachypodium and rice orthologoues - Download PDF. File Name: csc2cropsci2016040234-sup-0001.pdfResource Description: Brachypodium and rice candidates for Wsm2 orthologous to the colinear region in wheat chromosome 3B that were identified are presented.