Abstract: ESD (esterase D), GLO1 (glyoxalase I), and GPT (glutamate pyruvate transaminase) are human erythrocytic isoenzymes and have previously been applied in forensic medicine caseworks. The molecular bases of the polymorphic gene expression products have been demonstrated to be because of SNPs in respective coding regions. However, it has not been revealed whether the SNPs conferring the polymorphisms to the aforementioned erythrocytic isoenzymes could be simultaneously detected by using a simple PCR method. In this study, we used mismatched primers to simultaneously amplify three common isoenzyme loci so that all amplified products contained the same Hph I cleavage sites. The products were then digested with Hph I and electrophoretically separated and stained so that alleles were identified. The accumulated values for the probability of discrimination power and excluding the probability of paternity to the aforementioned systems attained 90.41% and 41.72%, respectively, in the Chinese Han population. This assay could be extremely valuable for future forensic medicine practices.