Objective: To analyze the mutation characteristic of 39 short tandem repeat (STR) loci in paternity testing from the Han population in Jiangsu Province. Methods:?A total of 6,812 paternity testing cases accepted by Forensic Identification Institute in the First Affiliated Hospital with Nanjing Medical University were collected from January 2019 to December 2024. The GoldenEye? DNA Identity System was used to detect mutations in 39 STR loci, and statistical analyses were performed on mutation rates, mutation origin and steps. Results:?Among the 6,812 confirmed paternity testing cases, there were 1,680 trios and 5,132 duos. A total of 214 mutation events were observed, with an overall mutation rate of?2.52%. The highest mutation rate occurred in?D12S391 (0.3297%, 28/8,492), followed by?Penta E, FGA, D21S11, D18S51 and?D3S1358 with mutation rates exceeding?0.2000%. In the analysis of mutation origin, 5 cases were of uncertain origin, while 189 cases were paternal mutations and 20 cases were maternal mutations. The mutations rate of paternal origin was significantly higher than that of maternal origin" (P<0.001). Among the mutations, 203 (94.86%) cases were single-step mutation, with 9 cases in two steps, and 2 cases in three steps observed at FGA and D21S11 loci. Conclusion:? The overall mutation rate of 39 STR loci used in paternity testing was relatively high, and there were significant gender and regional differences, which need to be given more attention in the practice of paternity testing. Our findings provide data support for the STR mutation status from the Han population in Jiangsu Province and contribute to improve the accuracy of complex kinship identification.