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To evaluate the applicability of 23 autosomal STR loci (D10S1248, D11S4463, D12ATA63, D14S1434, D17S1301, D18S853, D1GATA113, D1S1627, D6S1017, D20S1082, D20S482, D17S974, D22S1045, D1S1677, D2S1776, D2S441, D3S4529, D4S2408, D9S1122D5S2500, D6S474, D18S51, D9S2157) included in DNA Typer™ 25 Kit for individual identification and parentage testing, allele frequencies and forensic efficiency parameters were first obtained from healthy, unrelated 506 Chongqing Tujia individuals. A total of 1012 alleles were identified in 23 STR loci, and allele frequencies ranged from 0.001 to 0.5761. The combined power of discrimination (CPD) and the combined power of exclusion (CPE) of the 23 STR loci were 0.999999999999999999999753 and 0.99999967, respectively. These results suggested that 23 autosomal STR loci could be used as an effective tool for forensic application in Chongqing Tujia population. Comprehensive comparisons were conducted based on the analysis of genetic distance, principal component analysis (PCA), multidimensional scaling plot (MDS), and phylogenetic tree to explore the interpopulation genetic relationship. Our results revealed that Chongqing Tujia keeps the more relatively genetic similarity with Hunan Han, Hubei Tujia, and Sichuan Han, which could be interpreted by that those populations were originated from the same ethnic ancestor or genetic communication were happened in adjacent areas. [ABSTRACT FROM AUTHOR]