Recent reports of variant (non-subtype B) HIV infections in US populations have raised concerns about the sensitivity of subtype B virus-based donor screening and diagnostic assays. This study was designed to determine the prevalence and genetic diversity of HIV subtypes in US blood donors over the last two decades. Three groups were studied: hemophiliacs infected by clotting factor concentrates in the early 1980s (n = 49), blood donors retrospectively identified as being seropositive in 1985 (n = 97), and blood donors identified as seropositive between 1993 and 1996 (n = 405). Subtype assignment was based primarily on heteroduplex mobility analysis (HMA) of HIV-1 env, with DNA sequence confirmation of selected specimens. HIV peptide-based EIA serotyping was used to rule out HIV-2 and group O infections and to serotype HMA-refractory specimens. Of 551 specimens, 535 (97%) were assigned subtypes; 532 (99%) of these were subtype B. Three postscreening donations (1%) were assigned non-B subtypes (2 A, 1 C). Two of these three donors were born in Africa; the third was born in the United States and reported no risk factors other than heterosexual activity. HMA distribution plots showed an increase in env diversity among HIV-1 group B strains over time. The results support the need for continued surveillance of HIV subtype diversity and ongoing validation of the sensitivity of HIV diagnostic assays to non-B subtype infections.