Background: Hepatitis B virus (HBV) infection is a serious global public health problem, with WHO Western Pacific and African region worst hit. The objective of this study was to determine the prevalence of HBV infection and its associated factors among pregnant women accessing Ante-Natal Care (ANC) at a tertiary hospital in Enugu State, South-East Nigeria. Methods: This was a prospective cohort study that involved all the women that attended ANC of a tertiary hospital in Enugu State Nigeria for a period of 12 months (June 2020-May 2021). Data was retrieved from the patient’s ante-natal cards and entered into a pro forma. Data was analysed using SPSS version 25 and variables were presented as frequencies, percentages, means, and standard deviation. Chi-square test was used to determine factors that affected the prevalence of HBV with the level of significance set at p ≤ 0.05. Results: All the ANC attendees in the hospital were tested for HBV with annual prevalence of 1.0%. The mean age of the women was 29.78±4.70 with a range of 16-46 years. Women aged 31 – 40 years, married and with tertiary education had the highest HBV prevalence. Occupation and parity were the only factors associated with the prevalence of HBV. Conclusion: The Prevalence of HBV among the pregnant women showed low endemicity.