Most accessions of Dasypyrum villosum (2n = 2x = 14), a tertiary gene pool of wheat, were resistant in previous tests with a collection of Pgt races, including TTKSK. In order to transfer novel resistance genes with minimum linkage drag into wheat, chromosome-specific markers linked to the resistance loci would be useful. However, the currently available SNP and sequence information for D. villosum is very limited. Hence, developing SNP markers and genotyping D. villosum will aid in transferring stem rust resistance genes to wheat. We used genotyping-by-sequencing (GBS) to sequence a complete set of Chinese Spring (CS)-D. villosum addition lines (1V#3 to 7V#3) as well as CS and several D. villosum accessions to map D. villosum SNPs on each chromosome. We have also produced TTKSK-resistant CS-D. villosum amphiploids using other D. villosum accessions assuming they could carry different stem rust resistance genes.