Cross-sectional studies were conducted to determine the prevalence of antibody to Trypanosoma cruzi, the agent of Chagas' disease among three populations in Belize. Specimens were tested using a second-generation enzyme-linked immunoassay (EIA). Confirmatory testing with three single-antigen EIAs and a radioimmunoprecipitation assay (RIPA) were performed. Five (0.5%) of 962 blood donors at the Belize City Hospital were reactive including four (6%) of 65 donors from countries known to be endemic for Chagas' disease and one (0.1%) of 861 from Belize (P < 0.001). Among 467 healthy members of the Belize Defense Force, none were reactive. The third group included workers and families primarily from other Central American countries living on a banana plantation in a rural area of the country. Twenty-seven (6.1%) of 442 sera were reactive. The prevalence was 5.3% of 75 < 15 years of age, 4.2% of 236 15–34 years of age, and 9.7% of 124 ≥ 35 years of age (P = 0.11, by chi-square for trend). The prevalence was similar in males (6.7% of 280) and females (5.8% of 154). The prevalence of those born in Belize (4 of 56, 7.1%) was similar with that of those born in El Salvador (9 of 110, 8.2%), Guatemala (6 of 117, 5.1%), and Honduras (8 of 129, 6.2%). Of the four persons with reactive sera who were born in Belize, the immigrant mother of one was also reactive, suggesting possible congenital transmission. Among 31 sera repeatedly reactive by EIA to T. cruzi that were further studied, 22 (71%) were reactive by at least two of three single-antigen confirmatory EIAs and 29 (94%) by RIPA. Additional studies should focus on the epidemiology of T. cruzi and ways to reduce risk of transfusion-related infections in Belize.