Background: Hepatitis B virus (HBV) infection is a significant global public health issue. Although infant immunoprophylaxis effectively reduces viral transmission, mother-to-child transmission during pregnancy, often associated with intrauterine infection, remains a major route of HBV infection. Objectives: The aim of this study was to determine the prevalence of maternal HBV infection, identify its associated factors, and measure viral load in Hepatitis B surface antigen (HBsAg)-positive pregnant women in Zahedan, Iran. Methods: This cross-sectional study included data from pregnant women who were referred to the Central Laboratory of Zahedan, Iran, for routine pregnancy tests between August and December 2019. Data were collected during the first prenatal visit along with HBsAg testing. Hepatitis B surface antigen testing was repeated for HBsAg-positive women at 24 - 28 weeks of gestation. If the patient remained HBsAg-positive, serum viral load was determined. Factors associated with HBV infection were identified using multiple logistic regression models. Results: A total of 1,474 pregnant women participated in this study. Among them, 24 (1.6%, 95% CI: 0.9% - 2.2%) were HBsAg-positive, with 17 (71%) of these patients being unaware of their infection. The mean viral load in infected women was (11.1 ± 3.2) × 107 IU/mL. Only 13.3% of pregnant women were vaccinated against HBV. Maternal tobacco use, family history of HBV infection, previous history of HBV infection, and history of nonalcoholic fatty liver disease were significantly associated with HBV infection (P < 0.05). Conclusions: The prevalence of HBV infection remains high among pregnant women, and vertical transmission poses a major challenge in the elimination of HBV infection in southeastern Iran. In addition to active-passive immunoprophylaxis, universal screening of all pregnant women for HBV infection and the use of peripartum antiviral therapy in HBsAg-positive pregnant women with high viral loads are warranted.