The aim of this study was to determine the frequencies and parameters of 20 STR loci among Kazakh, Hui and Han population in Shihezi area of Xinjiang. The 20 STR loci of unrelated individuals of three population in Shihezi were amplified by DNATyperTM21 Kit, and the amplification products were analyzed through 3500xL genetic analyzer. Genetic polymorphism data and genetic distance were analyzed by PowerStats v12, Arlequin v3.5, Phylip-3.695 software. 231 alleles were identified in Kazakh population with their F ranging from 0.000 8～0.500 8. Heterozygosity (H) ranged among 0.669～0.923, discriminant power (DP) ranged among 0.817～0.988, polymorphism information content (PIC) ranged among 0.60～0.92, and paternity exclusion (PE) ranged among 0.380～0.843, comulative probability of exclusion presented 0.999 999 999 in triplet setting (CEPtri) and 0.999 998 212 in duo sttuations (CEPduo), total probability of discrimination power (TDP) was within 1-7.60×10–26; 237 alleles were identified in Hui population with their F ranging from 0.000 8 ～0.533 4. H ranged among 0.833～0.928, DP ranged among 0.949～0.986, PIC ranged among 0.81～0.91, and PE ranged among 0.661～0.853, comulative probability of exclusion presented 0.999?999?996 in CEPtri and 0.999?996 725 in CEPduo, TDP was within 1-4.77×10–25; 253 alleles were identified in Han population with their F ranging from 0.000?8～0.541?0. H ranged among 0.829～0.943, DP ranged among 0.958～0.986, PIC ranged among 0.83～0.91, and PE ranged among 0.655～0.884, comulative probability of exclusion presented 0.999?999?996 in CEPtri and 0.999?995?459 in CEPduo, TDP was within 1-1.15×10–24. The genetic distance was calculated by Phylip-3.695 softwarit, and was relatively far apart between Kazakh and Hui, Han, whereas relatively close between Han and Hui. The results of this work provided reference data for human population genetics and forensic DNA research in this area.