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Understanding genetic variability in existing wheat accessions is critical for collection, conservation and use of wheat germplasms. In this study, 138 Chinese southwest wheat accessions were investigated by genotyping using two resistance gene makers (Pm21 and Yr26) and DArT-seq technique. Finally, about 50% cultivars (lines) amplified the specific allele for the Yr26 gene (Gwm11) and 40.6% for the Pm21 gene (SCAR1265). By DArT-seq analysis, 30,485 markers (6486 SNPs and 23999 DArTs) were obtained with mean polymorphic information content (PIC) value 0.33 and 0.28 for DArT and SNP marker, respectively. The mean Dice genetic similarity coefficient (GS) was 0.72. Two consistent groups of wheat varieties were identified using principal coordinate analysis (PCoA) at the level of both the chromosome 6AS and the whole-genome, respectively. Group I was composed of non-6VS/6AL translocation lines of different origins, while Group II was composed of 6VS/6AL translocation (T6VS/6AL) lines, most of which carried the Yr26 and Pm21 genes and originated from Guizhou. Besides, a model-based population structure analysis revealed extensive admixture and further divided these wheat accessions into six subgroups (SG1, SG2, SG3, SG4, SG5 and SG6), based on their origin, pedigree or disease resistance. This information is useful for wheat breeding in southwestern China and association mapping for disease resistance using these wheat germplasms in future. [ABSTRACT FROM AUTHOR]