Infection with human T‐lymphotropic virus type 1/2 (HTLV‐1/2) is a major health problem. HTLV‐1/2 infection is endemic in Chile but representative donor prevalence data are lacking. Data on all blood donors in a large network of Chilean blood centers were examined during 2011–2013. Screening of HTLV‐1/2 antibodies were measured by enzyme immunoassay (EIA) at all blood banks. Blood samples with anticoagulants from initially reactive blood donors were analyzed by serological confirmation tests (immunofluorescence or recombinant immunoblot) at the HTLV National Reference Laboratory of the Public Health Institute of Chile. Additionally, detection of HTLV‐1 and HTLV‐2 provirus in peripheral blood mononuclear cells (PBMCs) was performed in all blood donors as confirmatory test. Prevalence rates were calculated. Among 694,016 donors, 706 were seropositive for HTLV‐1 (prevalence, 1.02 cases per 1,000; 95% confidence interval [CI], 0.94–1.09), and 97 were seropositive for HTLV‐2 (prevalence, 0.14 cases per 1,000; 95%CI, 0.11–0.17). Prevalence of HTLV‐1 differed considerably by region, from 0.51 to 1.69 per 1,000. Prevalence of HTLV‐2 was similar across the country (0.12–0.16). HTLV‐1 prevalence was associated with female sex, older age, and residence in the north of Chile. HTVL‐2 prevalence was associated with older age. The HTLV‐1 prevalence among Chilean blood donors was relatively high and could be reduced by improving donor recruitment and selection in high prevalence areas. Blood center data may contribute to surveillance for HTLV‐1 and HTLV‐2 infections. J. Med. Virol. 88:1067–1075, 2016. © 2015 Wiley Periodicals, Inc.