BACKGROUND: Genetic variations of human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) can affect diagnostic assays and therapeutic interventions. Recent changes in prevalence of subtypes/genotypes and drug/immune-escape variants were characterized by comparing recently infected vs more remotely infected blood donors. METHODS: Infected donors were identified among approximately 34 million US blood donations, 2006-2009; incident infections were defined as having no or low antiviral antibody titers. Viral genomes were partially sequenced. RESULTS: Of 321 HIV strains (50% incident), 2.5% were non-B HIV subtypes. Protease and reverse transcriptase (RT) inhibitor resistance mutations were found in 2% and 11% of infected donors, respectively. Subtypes in 278 HCV strains (31% incident) yielded 1a>1b>3a>2b>2a>4a>6d, 6e: higher frequencies of 3a in incident cases vs higher frequencies of 1b in prevalent cases were found (P = .04). Twenty subgenotypes among 193 HBV strains (26% incident) yielded higher frequencies of A2 in incident cases and higher frequencies of A1, B2, and B4 in prevalent cases (P = .007). No HBV drug resistance mutations were detected. Six percent of incident vs 26% of prevalent HBV contained antibody neutralization escape mutations (P = .01). CONCLUSIONS: Viral genetic variant distribution in blood donors was similar to that seen in high-risk US populations. Blood-borne viruses detected through large-scale routine screening of blood donors can complement molecular surveillance studies of highly exposed populations.