Knowledge of genetic diversity and population structure is essential for the effective use of wheat germplasms resistant to stripe rust in southwest China in breeding programs. In this study, DArT-seqTM based on genotyping by sequencing (GBS) approach was used to screen a collection of 134 common wheat varieties from the whole genome, and 6919 informative single nucleotide polymorphism (SNP) markers were obtained. The polymorphism information content (PIC) of SNP markers was in the range from 0.01 to 0.50, with the average of 0.32. SNP-based genetic similarity (GS) value among 134 wheat accessions ranged from 0.51 to 0.98, with the average of 0.61. The unweighted pair-group method with arithmetic mean (UPGMA) dendrogram indicated that the wheat accessions were clustered into five groups according to their origins and pedigrees. Two clear groups (Group I and Group II) were identified using principal-coordinates analysis (PCoA) of the whole-genome level. Group I is highly clustered with no obvious separation of cultivars (lines) originating from different geographic regions, most of them carried Yr9 locus; Group II was mainly composed of varieties from Guizhou province, most of them carried Yr26 locus. Our results provide valuable information of genetic diversity for stripe rust resistant wheat germplasms in Southwest China, which will facilitate wheat breeding for resistance to strip rust in our country.