We surveyed human immunodeficiency virus (HIV) subtype distribution from peripheral blood mononuclear cells (PBMCs) collected in 1995 from 24 HIV-1-infected Kenyan residents (specimens from predominantly male truck drivers and female sex workers near Mombasa and Nairobi). Processed lysates from the PBMC samples were used for env amplification, directly sequenced, and analyzed by phylogenetic analysis. Envelope amplification products were also used for analysis in a polymerase chain reaction (PCR)-based assay, called the combinatorial melting assay (COMA). Results of the two tests were compared for assignment of subtype for this Kenyan cohort. The COMA, a PCR capture technique with colorimetric signal detection, was used with HIV reference subtype strains as well as regional (East Africa) HIV strains for subtype identification. Performance of the COMA was at 100% concordance (24 of 24) as compared with DNA sequencing analysis. Phylogenetic analysis showed 17 isolates to be subtype A, 3 subtype D, and 4 subtype C viruses. This may represent an increase in subtype C presence in Kenya compared with previously documented reports. The COMA can offer advantages for rapid HIV-1 subtype screening of large populations, with the use of previously identified regional strains to enhance the identification of local strains. When more detailed genetic information is desired, DNA sequencing and analysis may be required.