Background &Objectives: Hepatitis B is a viral disease that often appears with the symptoms of liver inflammation. About 35 percent of the population had contact with Hepatitis B viruses. Given the lack of comprehensive information on prevalence of the disease among pregnant women as well as the concern on virus transmission from infected mothers to fetus during the pregnancy this research aimed to determine HBsAg prevalence and characterize its associated factors in pregnant mothers referred to Ardabil district health centers. Methods: In a cross sectional study all the 9321 pregnant women referred to Ardabil urban and rural Health Centers, Health Posts, and Health Houses were investigated in 2009. Experimental examinations of serum samples were performed using Elisa serology method and other information required were gathered by reviewing pregnant women health care records and analyzed using SPSS software. Results: Serum samples of 159 pregnant women (1.7%) were positive for HBsAg. The highest percent of research units (89.3%) were in age group of 18-35. The average number of pregnancy was 1.87±1.103. Significant differences were observed between hepatitis B prevalence and maternal education level, spouse job, age of pregnancy, and place of residence. Conclusion: Ardebil city is among the areas with intermediate prevalence of positive hepatitis B surface antigen and living in rural area is a main risk factor associated with the prevalence of positive cases in the region.