Proso millet is an important allotetraploid cereal crop; however, it is the least studied species of the Poaceae family, as it is an under-utilized crop. Genomic resources for proso millet are very limited compared to major crops. An understanding of the genetic relationships among germplasm resources is important for future breeding endeavors. In the present study, simplesequence repeat (SSR) markers were employed to assess the polymorphism and genetic diversity of 100 millet accessions from different countries, which were tested in the dry steppe zone conditions of the Akmola region from 2020 to 2022. The use of 20 SSR markers detected a total of 47 alleles, with an average allele number of 2.35 per locus among these proso accessions. Nine of them were polymorphic among the genotypes, which suggests that these SSR markers can be used for genetic studies. The results showed a moderate level of polymorphism information content (PIC) that averaged at 0.424, ranging from 0.125 to 0.795. The markers SSR-67, SSR-82, SSR-85 and SSR-109 showed high PIC values of 0.536, 0.756, 0.795 and 0.758, respectively. Markers SSR 85 and SSR 86 significantly correlated to agronomic traits, such as productive tillering (PT) and grain yield (GY). The genetic structure, UPGMA cluster and PCoA assay indicated that the accessions that originated from Central Asia had higher genetic diversity. Based on structure (K = 3), all the accessions were divided into three groups, where the gene pool that originated from Central Asia wasdetected in all three clusters. Based on a principal component analysis (PCA), the accessions of Central Asian origin were genetically closer to the North Asian group.