Background: Hepatitis B virus (HBV) infection is a serious public health problem in worldwide and the major cause of morbidity and mortality. Globally, 350 million people are chronically infected with hepatitis virus and more than 68,600 people die of HBV infection. Pregnant mother infected with Hepatitis B virus has a high rate of vertical transmission, causing adverse fetal and neonatal outcome. Understanding the epidemiology of Hepatitis virus could have paramount importance to take appropriate preventive measures. The world health organization recommends screening of all pregnant mothers. However, this practice is poor currently in Ethiopia. Hence, this study aims to assess the prevalence of hepatitis B virus infection and associated factors. Methods: Cross-sectional study was conducted in selected governmental health facilities of Harar city from March to April 2017. Systematic random sampling technique was used to select 326 study participants. Data were collected using pretested interviewer administered questionnaire. Serum was withdrawn from each study participants and used to detect hepatitis B Antigen surface using an enzyme linked immunosorbent assay test kit. Descriptive, bivariate and multivariate logistic regression analysis was done to identify independent predictors of hepatitis B virus infection after controlling for confounding variables. Result: Prevalence of hepatitis B virus among pregnant mothers was found to be 6.3%. In multivariate analysis; blood transfusion [AOR=9.7, 95% CI (1.6, 57.5)], history of surgical procedure [AOR=4.1, 95% CI (1.1, 16.8)], history of sexually transmitted infection [AOR=10.1, 95% CI (2.9, 34.7)], being HIV positive [AOR=13.1, 95% CI (1.8, 95.6)] and history of tooth extraction [AOR=5.5, 95% CI (1.4, 22.1)] were independent predictors of HBV infection. Conclusion: HBV infection is intermediate endemicity in the study area. Higher prevalence of HBV infection was identified among mothers with history of blood transfusion, history of surgical procedure, history of STI, HIV positive and history of tooth extraction.