DNA fragments of HIV-1 env gene were amplified by nested-PCR from 17 uncultured peripheral blood mononuclear cells (PBMCs) obtained from HIV-1 seropositive intravenous drug users (IDUs) in Ruili city of Yunnan Province. The C2-V3 region (about 450 bp) of them were sequenced. Sequence analysis showed that there exists two HIV-1 subtypes, B and C, with 5.8% and 2.2% gene divergence inside each subtype. The 12 subtype B strains, were closely related to those found in Thailand, Myanma and Ruili city of Yunnan, and the nucleotide sequence divergence between them ranged from 4.4% to 4.9%; meanwhile, the 5 subtype C strains were most close to those found in India as well as Ruili city, all with a genetic distance of 1.9%. The small divergence among Ruili HIV-1 subtype C strains suggests a recent epidemic. The analysis of V3 loop amino sequence of 12 subtype B HIV-1 reveals that V3-tip motif of 6 samples (50%) is GPGQ and that of 3 samples (25%) is GPGR. In addition, the codon of arginine (R) of all the strains is CGA instead of AGA. This result is in accordance with our previous hypothesis that there is a drift in vivo from GPGR to GPGQ motif on the tipof V3-loop of HIV-1 subtype B strain in this arm with the elapse of time.