The prevalence of hepatitis B virus (HBV) markers was studied in pregnant women attending antenatal clinics in Zambia. A total of 2,098 pregnant women were recruited into the study at three urban health centres in Lusaka, the capital city and four district hospitals in rural areas of different provinces of Zambia. The overall prevalence of HBsAg was 6.5% (137/2,098), and HBeAg was present in 16.1% (22/137) of those positive for HBsAg. Antibody positive rate (HBsAb and/or HBcAb) was 51.3% in randomly selected HBsAg negative samples. HBsAg positive rate varied between 3.3% and 13.6% in each study sites. Prevalence for both HBsAg and antibodies to HBV were significantly higher in rural areas (district hospitals) than in urban areas (urban health centres in Lusaka). These data show that although HBV is endemic in Zambia, the prevalence varies from region to region.