ABSTRACT: In the present study, we investigated the genetic characteristics of 25 Y-chromosomal and 15 autosomal short tandem repeat (STR) loci in 305 unrelated Han Chinese male individuals from Liaoning Province using AmpFISTR® Yfiler® Plus and IdentifilerTM PCR amplification kits. Population comparison was performed between Liaoning Han population and different ethnic groups to better understand the genetic background of the Liaoning Han population. For Y-STR loci, the overall haplotype diversity was 0.9997 and the discrimination capacity was 0.9607. Gene diversity values ranged from 0.4525 (DYS391) to 0.9617 (DYS385). Rst and two multi-dimensional scaling plots showed that minor differences were observed when the Liaoning Han population was compared to the Jilin Han Chinese, Beijing Han Chinese, Liaoning Manchu, Liaoning Mongolian, Liaoning Xibe, Shandong Han Chinese, Jiangsu Han Chinese, Anhui Han Chinese, Guizhou Han Chinese and Liaoning Hui populations; by contrast, major differences were observed when the Shanxi Han Chinese, Yunnan Bai, Jiangxi Han Chinese, Guangdong Han Chinese, Liaoning Korean, Hunan Tujia, Guangxi Zhuang, Gansu Tibetan, Xishuangbanna Dai, South Korean, Japanese and Hunan Miao populations. For autosomal STR loci, DP ranged from 0.9621 (D2S1338) to 0.8177 (TPOX), with PE distributing from 0.7521 (D18S51) to 0.2988 (TH01). A population comparison was performed and no statistically significant differences were detected at any STR loci between Liaoning Han, China Dong, and Shaanxi Han populations. The results showed that the 25 Y-STR and 15 autosomal STR loci in the Liaoning Han population were valuable for forensic applications and human genetics, and Liaoning Han was an independent endogenous ethnicity with a unique subpopulation structure.