A comprehensive seroepidemiologic study was conducted in two Red Cross regions (Los Angeles and Miami) to determine the prevalence of Trypanosoma cruzi antibodies in at-risk blood donors, to identify additional risk factors, and to assess the likelihood of transmitting T. cruzi by transfusion. At-risk and control donors were stratified by a broad risk question, tested for T. cruzi antibodies, and if confirmed as seropositive, enrolled in case-control and lookback investigations. A total of 299,398 donors were queried; 23,978 at-risk and 25,587 control donations were tested, and T. cruzi antibodies were confirmed in 34 donors (33 and 1, respectively). Seropositive donors shared one risk factor; birth/extensive time in a T. cruzi-endemic area. Lookback studies identified 11 recipients, all negative for T. cruzi antibodies. Screening strategies that use a question are unlikely to identify all seropositive donors. The lack of definitive data on the risk of transmission by transfusion indicates additional studies of donors and recipients are needed.