OBJECTIVE To study human ABO genotyping by means of multiplex PCR and restriction fragment length polymorphism. METHODS Two specific fragments of ABO gene were simultaneously amplified in a single tube, and then the double restriction digestion with RE Kpn I and Alu I was performed in the same tube. The amplified products were analyzed by PAGE and silver staining. A total of 125 Han unrelated individuals living in Wuhan were genotyped. RESULTS Six genotypes of ABO were detected and the distribution was in good agreement with Hardy-Weinberg equilibrium. CONCLUSION The results demonstrate that PCR-RFLP based approach is convenient, reliable and should be of value in forensic application.