It is estimated that 15-20 millions of people are infected with human T-cell lymphotropic virus 1/2 (HTLV-1/2) worldwide, and 20-30% of them are Latin Americans. The seroprevalence rates vary according to geographic area, socio-demographic composition and individual risk behaviors. The impact of HTLV-1-associated diseases on the community, as well as the increasing concerns about blood transfusion safety due to infectious transmitted agents, has required mandatory screening assays of blood donors as an effective preventive strategy in HTLV-1 transmission. The aim of this study was to estimate the seroprevalence of HTLV-1/2 in blood donors from Valdivia, southern Chile during the first year of mandatory screening of HTLV by Chilean government. Blood samples were collected from blood donors between May 2009 and 2010 at the Regional Hospital of Valdivia, Chile. Serum samples were screened for antibodies against HTLV-1/2 by enzyme-linked immunosorbent assay MUREX HTLV I + II Abbott. Among 6,237 samples tested by serological assay, 28 samples were reactive; nevertheless, confirmatory techniques indicate that only 15 samples were positive to HTLV-1. Our study shows HTLV-1 seroprevalence of 0.24%, indicating a similar seroprevalence associated with blood donors than previous reports for our country.