The retroviruses human immunodeficiency virus (HIV)-1/2 and human T-cell leukemia virus (HTLV)-I/II share modes of transmission, suggesting that efforts to monitor the current HIV-1 epidemic in Switzerland should be complemented by assessment of HTLV-I/II prevalence. This study presents an updated evaluation of HTLV-I/II infection among groups within the Swiss population polarized towards either low or increased risk of infection. Archived serum and peripheral blood mononuclear cell (PBMC) samples were examined for evidence of HTLV-I/II infection by enzyme-linked immunosorbant assay (ELISA), type-specific Western blot, type-specific polymerase chain reaction (PCR), DNA sequence analysis, and virus culture. Among blood donations obtained from low-risk Swiss donors, we report a complete lack of HTLV-II infection and the occurrence of HTLV-I infection limited to a prevalence of 0.079 per 100,000 (1/1,266,466). Among high-risk HIV-positive persons and HIV-negative persons at increased risk of HIV-infection, we report a focus of HTLV-I and HTLV-II infection at prevalence rates of 62 per 100,000 (1/1,620) and 309 per 100,000 (5/1,620), respectively. The finding of low HTLV-I/II prevalence among Swiss blood donors and containment of HTLV-I/II infection within known risk-groups does not support initiation of HTLV-I/II screening for Swiss blood, tissue, and organ donations.