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Background: Hepatitis B virus (HBV) infection is one of the most common health problems worldwide and is associated with high mortality and heavy economic burdens. The aim of this study was to determine the prevalence of hepatitis B surface antigen (HBsAg) and associated risk factors among pregnant women attending antenatal clinics at Adigrat General Hospital in Northern Ethiopia. Methods: An institutional-based cross-sectional study was conducted from January to March 2024 among 385 pregnant women. Participants were selected through a systematic random sampling method. Socio-demographic and associated factor data were collected using a structured questionnaire, and 5 mL blood samples were collected. The data were subsequently entered into EPI Info and analyzed using SPSS version 25. Descriptive statistics were computed. Bivariate and multivariate regression analyses were employed to measure associations, and values < 0.05 were considered to indicate statistical significance. Results: The overall seroprevalence of HBsAg among the 385 study subjects enrolled was 10.4% (n = 40). HBsAg was common in all age groups. The prevalence of infection was greater in those who had a history of multiple sexual partners (22.7%), early piercing (13.4%), or abortion (27.6%), as was the history of delivery (26.3%) and genital mutilation (35.7%). According to multivariate logistic regression, patients were unmarried (AOR 8.57; 95% CI 3.20 - 22.93), illiterate (AOR 12.06; 95% CI 3.07 - 47.33), had a history of ear piercing (AOR 5.66; 95% CI 1.65 - 19.45), a history of abortion (AOR 8.16; 95% CI 3.18 - 20.95), a history of home delivery (AOR 6.69; 95% CI 1.26 - 35.53), and a history of genital mutilation (AOR 9.77; 95% CI 2.64 - 36.18) for acquiring HBV infection compared to their counterparts. Conclusions: The results showed that HBV was highly prevalent in our study area. Being unmarried, having a low educational level, having an ear piercing, having an abortion, having a home delivery, and having genital mutilation were significantly associated with HBV infection. Therefore, these findings suggest that health education programs should be provided to the community to increase awareness among mothers. [ABSTRACT FROM AUTHOR]