Hepatitis B virus (HBV) infection is a serious public health problem in sub-Saharan Africa pregnant women. HBV Infection has high tendency of vertical transmission and have adverse effect on both the mother and child. However, there is no evidence on prevalence of hepatitis B virus among pregnant women in Wolaita Zone. Therefore, this study aims to determine prevalence and associated factors of hepatitis B virus infection among pregnant attending Antenatal clinics of public Hospitals of Wolaita Zone. An institution based cross sectional study was conducted among pregnant women attending antenatal clinics of Wolaita Zone from October-November, 2018. Systematic random sampling was used to get respondents. A pretested, structured questionnaire was used to collect socio-demographic characteristics and other variables. In addition, 5 ml of venous blood was collected from each study participants and serum was tested for Hepatitis B surface antigen. Data was entered to Epidata 3.1 version and exported Statistical Package for Social Sciences Version 20.0 for descriptive and logistic regression analysis. All variables in bivariate analysis with p<0.25 were taken to multivariable analysis. P-value and Odds ratio with 95% CI was used to measure the presence and strength of the association respectively. The prevalence of Hepatitis B surface Antigen among pregnant women was 49(7.3%). History of multiple sexual partners (AOR = 2.675, 95%CI = 1.107-6.463), surgical procedure (AOR = 3.218, 95%CI = 1.446-7.163), genital mutilation (AOR = 2.72, 95% CI = 1.407-5.263), and tooth extraction (AOR = 2.049, 95%CI = 1.061-3.956) were statistically associated with HBV. Intermediate endemicity of Hepatitis B Virus (7.3%) was observed among mothers attending antenatal clinics of Wolaita Zone. History of tooth extraction, history of surgical procedure, history of genital mutilation and history multiple sexual partners were factors associated with acquisition of Hepatitis B Virus infection. Therefore, we recommend that the health education programs should be done to avoid traditional and non-sterile tooth extraction methods, female genital mutilation and avoiding having multiple sexual partner and its consequences to community and to raise the awareness of mothers attending antenatal clinics. Facilities should strictly follow sterile procedures in every surgical procedure.