Extensive transmission of human immunodeficiency virus type 1 (HIV-1) in Thailand began in 1988, resulting in an estimated 800,000 cumulative infections by 1994, During 1994 and 1995, we collected blood specimens from 215 asymptomatic HIV-1-infected people with various risk behaviors from nine locations in all four regions of Thailand, HIV-1 subtypes and genetic heterogeneity were determined for 214 strains by a combination of direct DNA sequencing (n = 95), subtype-specific oligonucleotide probe testing (n = 201), and V3-loop peptide enzyme immunoassay (PEIA) (n = 214), All strains were either env subtype E (175; 81.8%) or B (39; 18.2%), Of the subtype B isolates, 37 (94.9%) were B' and 2 (5.1%) were more typical North American-like B strains (most subtype B strains in Thailand are part of a distinct subcluster within the subtype B branch on phylogenetic trees, termed B'; formerly Thai B or B-B) Of 149 viruses from people with sexual risk behaviors from all regions, 146 (98.0%) were subtype E, Of 65 viruses from injecting drug users (IDUs), 29 (44.6%) were subtype E and 36 (55.4%) were subtype B, including 35 B' strains, There was regional variation in the proportions of subtypes E and B' among IDUs, The intrasubtype nucleotide divergence within the V3 and flanking regions of the env gene (mid-C2 to the start of the V4 region) was low (5.7% for subtype E and 3.1% for subtype B') compared with other HIV-1 group M subtypes from different countries, These findings of two subtypes with low heterogeneity indicate that Thailand may be a desirable setting for evaluating candidate HIV-1 vaccines, The mix of subtype E and B' strains among IDUs also offers the opportunity to study phenotypic differences between the two subtypes.