Objective To study the genetic polymorphism of 20 autosomal short tandem repeats(STR)loci in Yunnan Han population. Methods The 20 STR loci(D3S1358,D1S1656,D6S1043,D13S317,Penta E,D16S539,D18S51,D2S1338,CSF1PO,Penta D,TH01,vWA,D21S11,D7S820,D5S818,TPOX, D8S1179,D12S391,D19S433 and FGA)which were included in the PowerPlexR21 System kit were genotyped in 1085 unrelated Han individuals living in Yunnan province using multiplex amplication. PCR products were separated and analyzed by the AB 3130 automatic genetic analyzer and GeneMapper ID v3.2 software. Forensic parameters of each locus were calculated by Modified-Powerstates software. Results All the studied loci except for TH01 and TPOX were highly polymorphic. The observed heterozygosity(Ho)ranged from 0.6130 to 0.8743. Match probability(PM)ranged from 0.0179 to 0.2030. Power of discrimination(DP)ranged from 0.7970 to 0.9821. Probability of exclusion(PE)ranged from 0.3067 to 0.7432. Paternity index(PI)ranged from 1.2919 to 3.9766. Polymorphism information content(PIC)ranged from 0.5598 to 0.8958. No deviation of the Hardy-Weinberg equilibrium was observed. Conclusion The studied 20 STR loci were highly polymorphic in Yunnan Han population and could be used in forensic individual identification and paternity testing practice.