A seroprevalence survey of hepatitis B virus (HBV) markers was conducted among health care workers in Belize to help determine the epidemiology of hepatitis B and to determine if screening before immunization might lower vaccine costs. Of the 330 workers tested, 94 (29%) were positive for antibody to HBV core antigen (anti-HBc) and three (1%) had HBV surface antigen. The presence of anti-HBc increased significantly with age from 12% in those 18–24 years old to 52% in those ≥ 50 years old. The rate was 17% of 48 men compared with 30% of 282 women (P = 0.05). Rates increased with years of medical service and were higher among nurses (69 of 228; 30%) and nonprofessional staff (15 of 44; 34%) than among physicians (0 of 20). The presence of anti-HBc also differed significantly among ethnic groups: Mestizo, 4%; Creole, 33% and Garifuna, 57%. Rates differed by district ranging from 3% in a northern district (mostly Mestizo) to 67% in a southern district (mostly Garifuna). Parenteral exposure to hepatitis B through needle stick injuries and blood transfusions was not associated with anti-HBc. Multiple logistic regression analysis confirmed ethnicity, district of residence, and age as the best predictors of anti-HBc in health care workers. Cost analysis suggests that because of regional differences in exposure, testing of health care workers for anti-HBc in the Belize and Stann Creek districts in southern Belize before hepatitis B immunization would result in vaccine program cost savings.