In this study, a total of 484 unrelated healthy individuals and 359 two-generation families in the Han population in Shanghai, China were successfully analyzed with the Investigator HDplex Kit (Qiagen, Hilden, Germany). Hardy–Weinberg equilibrium tests demonstrated no significant deviation from expected values (p > 0.05) for the 12 autosomal STRs included in this kit. A total of 173 alleles were detected in 12 autosomal STR loci; the largest number of alleles (32) was detected for the SE33 locus. The combined power of discrimination was 0.99999999992. The combined mean exclusion chance in duo cases was 0.99982, whereas the combined mean exclusion chance in trio cases was 0.9999986. The average mutation rate across all loci was 0.0023 (95% confidence intervals: 0.0013–0.0039). These results suggest that the 12 autosomal STR loci can provide highly informative polymorphic data for paternity testing and forensic identification in the Han population in Shanghai, China.