To genetically characterize HIV-1 strains in injecting drug users (IDU) in Bangkok, Thailand in 1994, and compare these with strains found earlier in Thai IDU; such information is essential for HIV-1 vaccine development and evaluation. Peripheral blood mononuclear cells were collected from 84 IDU attending 14 drug treatment clinics in Bangkok in 1994. DNA was amplified using a nested polymerase chain reaction (PCR) procedure and sequenced directly (without cloning) from the PCR products. The V3 and flanking regions (345 nucleotides) of the env gene were analyzed using a neighbor-joining tree. Only one (1%) strain was a typical subtype B virus, 69 (82%) were genetically distinct subtype B' viruses (Thai B), and 14 (17%) were subtype E strains (Thai A). Persons with recently acquired infection were more likely to have subtype E viruses (P < 0.001) than those in our 1991 survey, who were more likely to have subtype B' viruses. Pairwise intra-subtype differences within subtypes E and B' were 5.3 and 4.3%, respectively, compared with 3.4 and 3.5% among strains collected in 1991 in Thailand. The genetic diversity within subtypes B' and E in Thailand and the proportion of new infections due to subtype E viruses among Bangkok IDU are increasing significantly. These data highlight the importance of monitoring the molecular epidemiology of HIV-1 in populations being considered for HIV-1 vaccine trials.