Production of spring wheat, the major crop in Mongolia, accounts for 98% of the cultivated area. Understanding genetic variability in existing gene bank accessions is critical for collection, conservation and use of wheat germplasms. To determine genetic diversity and population structure among a representative collection of Mongolian local wheat cultivars and lines, 200 wheat accessions were analyzed with 15 SSR markers distributed throughout the wheat genome. A total of 85 alleles were detected, with three to five alleles per locus and a mean genetic richness of 5.66. Average genetic diversity index was 0.69, with values ranging from 0.37-0.80. The 200 Mongolian wheat accessions were mainly divided into two subgroups based on structure and phylogenetic analyses, and some phenotypes were divergent by the subgroups. Results from this study will provide valuable information for conservation and sustainable use of Mongolian wheat genetic resources.