Background Hepatitis B virus (HBV) is one of the major threats to global health, affecting millions of people each year and causing morbidity and mortality. The risk of acquiring chronic HVV infection varies with age, with 90% of infections acquired during the perinatal period. Despite many studies, there is little evidence of this virus in the Borena Zone. Objectives This study was done to assess seroprevalence HBVinfection and associated factors among pregnant women attending antenatal care at selected Borena Zone public hospitals from June 1 to September 30, 2022. Methods A cross-institutional study was conducted on 368 randomly selected pregnant women who received antenatal care at Yabelo General Hospital and Moyale Primary Hospital. Data on sociodemographic and HBV-related factors were collected using a structured questionnaire. A 5 mL blood sample is then taken and tested using a diagnostic method, the enzyme-linked immunosorbent assay. Finally, data were entered using Epidata version 3.1 and exported to SPSS version 25 and Stata version 14 for analysis. Independent predictors were identified using logistic regression analysis and P < .05 was considered statistically significant. Results The prevalence of HBV infection was 21 (5.7%) (95% confidence interval [CI], 3.74-8.61). History of hospitalization (adjusted odds ratio [AOR] =  3.44, 95% CI, 1.07-11.09), traditional tonsillectomy (AOR =  4.16, 95% CI, 1.29-13.40), history of sexually transmitted infection (AOR = 5.99, 95% CI, 1.81-19.85), human immunodeficiency virus (HIV) (AOR = 8.28, 95% CI, 1.66-41.41), and alcohol drinking (AOR = 5.08, 95% CI, 1.49-17.31) were independent predictors of HBV infection. Conclusions The results of this study indicate a moderate prevalence of HBV in selected public hospitals in the Borena Zone. History of hospitalization, traditional tonsillectomy, sexually transmitted infections, HIV, and alcohol use was significantly associated with HBV infection. Therefore, there is a need to conduct health education and more community-based research on disease transmission routes.