Objective：To analyze the mutation characteristics 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 of Nanjing Medical University were collected from January 2019 to December 2024. The GoldenEyeTM DNA Identity System was used to detect mutations in 39 STR loci，and statistical analyses were performed on mutation rates，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（0.329 7%，28/8 492）， followed by Penta E，FGA，D21S11，D18S51，and D3S1358 with mutation rates exceeding 0.20% . Paternal mutations accounted for 189 cases，maternal for 20 cases，and 5 cases were of undetermined origin，with paternal mutations significantly outnumbering maternal ones（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 STR mutation rate in paternity testing is relatively high（2.52%），showing gender and regional variations，which warrants attention in forensic practice. Our findings provide data support for the STR mutation status from the Han population in Jiangsu province and contribute to improving the accuracy of complex kinship identification.