Background: Knowledge of factors associated with seroprevalence of Hepatitis B in pregnancy is important in informing policies towards prevention of vertical transmission of Hepatitis B Virus (HBV) infection, since in-utero and perinatal infection lead to chronic carrier state with severe long-term sequela. In addition, adequate and timely immunization plays a major role in the prevention of transmission. Objective: To determine the factors associated with HBs Ag seroprevalence in pregnant women from various geographical regions in Kenya. Methods and Subjects: This was a cross-sectional survey amongst all pregnant women attending antenatal clinics in Kenyatta National Hospital and 8 other hospitals from different regions of Kenya in their third trimester of pregnancy among June 2001 to June 2002. For each pregnant woman, age, history of intravenous drug use, sexually transmitted disease (STD), liver diseases, alcohol intake, blood transfusion (BT) and presence of traditional scarification were documented. HBs Ag serology was assayed at the Kenya Medical Research Institute (KEMRI) laboratory. Results: A total of 2241 pregnant women were enrolled in the study, 2196 consented, of whom 205 (9.3%) tested positive for HBs Ag. A significant association was found between HBs Ag seroprevalence and traditional scarification (p = 0.029), history of blood transfusion (p = 0.0024) and alcohol intake (p = 0.05). There was no significant association between Hbs Ag seroprevalence and sexually transmitted disease (p = 0.64). It was not possible to evaluate the association of HBs Ag seroprevalence with history of hospitalization for any liver disease including Hepatitis A, B or C, history of intravenous drug use or contact with sex partHow to cite this paper: Gatheru, Z., Murila, F., Mbuthia, J., Okoth, F., Kanyingi, F., Mugo, F., Esamai, F., Alavi, Z., Otieno, J., Kiambati, H. ners previously hospitalised for any liver disease including Hepatitis A, B or C as the numbers with these attributes were small. Conclusions: We found 9.3% of pregnant women were HBs Ag positive, significantly higher than previously observed. The high seropositivity was associated with traditional scarification, history of blood transfusion and alcohol intake. Proper screening of blood transfusion products, immunization and control of scarification and alcohol use should be considered by health policy experts.