Polymorphic analysis of human phosphoglucomutase-3 (PGM(3)) has been carried out from the level of the gene product. Due to a weak zymogram, leading to ambiguity in phenotyping, information on the PGM(3) locus has rarely been reported. In this study, the missense mutation G1396A, confirmed to underlie common phenotypes of PGM(3), was identified by performing mismatched PCR-RFLP. Population data on the PGM(3) locus was also obtained for the first time in China. The allele frequency distribution was PGM(3)*1 = 0.625, PGM(3)*2 = 0.375, and no deviation from Hardy-Weinberg equilibrium was observed. The application of the information in both genetics and forensic medicine demonstrated that the polymorphism information content was 0.5163, heterozygosity 0.4872, power of discrimination 0.5986, and probability of paternity exclusion 0.1794. Polymorphic analysis of the locus at the DNA level will also provide significant data for disease susceptibility and linkage analysis.