Background: Sudan is a highly endemic country for hepatitis B virus (HBV). Screening for HBV during pregnancy may help to decide on appropriate antiviral therapy and the institution of steps to minimize vertical transmission to the newborns. Of the epidemiological studies carried in different regions of Sudan, few are available regarding pregnant women. In Darfur region such data is absent, so our aim was to evaluate seroprevalence of HBV among antenatal care attendants in Al Fashir town. Method: A cross sectional study was conducted between 2013 and 2015. Blood samples were collected from 900 pregnant women attending antenatal clinics in Al Fashir town. Plasma was separated and tested for markers of HBV (HBcAb, HBsAg, HBeAg, HBeAb) using ELISA. Sociodemographic, obstetrics and medical data were collected using structured questionnaires. Results: AntiHBc was detected in 46% and HBsAg was detected in 18% of study population. HBeAg and HBeAb frequencies were found to be 2.6% and 37.7% among positive HBsAg pregnant women. There was significant association between residence, income, occupation, bloodletting and ear piercing with HBV infection (P < 0.05). Conclusion: The results of the study suggest that HBsAg has a high prevalence among the pregnant women in Al Fashir town, North Darfur State. So, to minimize vertical transmission, antenatal women must be routinely screened for HBV.