Background: Hepatitis B virus infection is a major public health problem worldwide which is a major cause of morbidity and mortality. This study aimed to assess the prevalence of hepatitis B virus infection and associated factors among pregnant mothers in Gedio Zone, southern Ethiopia. Methods: Institutional based cross-sectional study was conducted in governmental and private health facilities in Gedeo zone from January to April 2019. The study participants were selected using stratified random sampling techniques. Eugene strip test was used to determine hepatitis B virus infection among pregnant mothers. The status of HIV was collected from the records. Other variables were collected from the mothers using interviewer administered questionnaires. Logistic regression was used for the analysis. Adjusted Odds Ratios and their 95% Confidence Interval were calculated to determine association between HBsAg Sero-positivity and various factors. A p-value less than 0.05 were considered as significant. The data was analyzed using the SPSS version 25 statistical software. Results: Prevalence of hepatitis B virus among pregnant mothers was 9.2% in Gedio Zone. Previous birth at health institution [AOR=4.4, 95% CI: 1.7, 11.2], blood transfusions [AOR=4.4, 95% CI: 1.8, 10.5], previous history of Hospital admission [AOR=3.3, 95% CI: 1.5, 7.5], ear piercing practice [AOR=5.7, 95% CI: 1.1, 29.0], current Gestational age [AOR=3.6, 95% CI: 1.2, 11.2], and HIV status of the mother [AOR=6.1, 95% CI: 1.3, 30.0] had statistical significant association with HBsAg Sero-positivity. Conclusions: Hepatitis B virus infection was found to have higher endemicity (9.2%) in the Gedio Zone. History of blood transfusion, hospital admissions, ear piercing, being HIV positive, gestational age and institutional delivery were significant predictors for HBsAg sero-positivity. Early initiation of antenatal care service that integrate awareness creation about the risks of hepatitis B infection and mother to child transmission of the disease must be implemented by the health facilities in Gedio Zone.