Abstract: Objective To establish a rational, detailed and feasible HIV-1 subtyping assay. Methods The viral loads of the plasma samples collected from men who have sex with men (MSM) infected with HIV in Nanchang were detected. The viral RNAs were extracted, and RT-PCR was performed in the Env and Gag region of these RNAs by using designed primers. The amplified cDNAs were sequenced. The sequences were aligned and the molecular phylogenetic trees of DNA in Env and Gag regions were constructed respectively. Based on these trees, the subtypes of HIV-1 were determined. The characteristics of amino acid sequences located in the V3-loop of Env region were analyzed. Results Among the three samples detected, the viral load of one sample was 43.9 copies/ml, and the viral load of other two were less than 20 copies/ml. All the strains from three samples belonged to CRF01_AE subtype. Conclusion The established HIV-1 subtyping assay was rational and detailed, which can provide some information for molecular epidemiological research in China.