To assess the molecular epidemiology of human immunodeficiency virus type 1 (HIV-1), a screening method was developed for identification of non-B subtypes from sequence data obtained for resistance testing. The method is based on the evaluation of the percentage of divergence of a given sequence from the reference B subtype HXB2. Analysis of 1720 reverse-transcriptase (RT) and 1824 protease sequences stored in a database allowed for the determination of a threshold level of divergence from HXB2 above which a non-B subtype could be unambiguously characterized regardless of the pattern of resistance mutations (>8.6% for RT; >10.8% for protease). This conclusion was validated by phylogenetic analysis of RT, protease, and env genes. Overall, 72 (4.2%) and 73 (4.0%) non-B sequences were identified in the RT and protease coding regions, respectively. This method allows for the rapid detection of non-B subtypes among retrospective, recent, and future RT and/or protease sequence databases.