To estimate the prevalence of HIV strains other than the predominant HIV-1B subtype in the U.S. blood donor population we genetically and serologically characterized HIV in infected blood donations collected throughout he United States from 1997 to mid-2000. Using a combination of DNA heteroduplex mobility and DNA sequence analyses of the env and gag regions of HIV-1 we determined that 285 of 312 infections were caused by HIV-1B and six by non-subtype B HIV-1 (four HIV-1C, one HIV-1AE, one HIV-1A). Genetic distances of greater than 14% in the envelope V3-V5 region of the four HIV-1C strains indicated that they did not share a recent common origin. HIV-1 group M, N, and O, and HIV-2 specific peptide serological testing of the 20 PCR-negative samples determined that one infection was caused by HIV-2 and none by HIV-1 group N and O. The major risk factor for infection with a non-HIV-1B strain was sex with an HIV-infected person from Africa although three of seven non-HIV-1B-infected subjects did not fit that category. For four of seven non-HIV-1B-infected subjects the subtype detected was consistent with the African country of origin of the infected person or of their sexual partner. The frequency of genetically confirmed non-subtype-B HIV infection in a geographically dispersed group of infected U.S. blood donors in 1977-2000 was therefore 2.0% (6/312).