Background: Anogenital colonization of group B streptococcus in pregnancy is common, which is identified as common cause of early onset neonatal disease. Most neonates acquire the disease through vertical transmission during labor and delivery. However, the prevalence of group B streptococcus colonization in pregnancy in this particular area is not studied. Objective: To assess prevalence of maternal anogenital group B streptococcus colonization and associated factors among pregnant mothers attending antenatal care at two referral hospitals in Bahir Dar city, Amhara region, Ethiopia. Methods: Institutional based cross sectional study design was conducted from March 1st to July 30th, 2019 on pregnant mothers attending antenatal care at TGSH and FHCRH b/n GA of 35-37weeks. Total of 195 pregnant mothers in the study with response rate of 97.5%. Data was collected with structured questionnaire and anogenital swab taken using cotton applicator, transported in charcoal medium and cultured on blood agar medium. Data entered to epi info 7, transported to SPSS version 23 and analyzed using both bivariate and multivariate analysis. Result: Among total of 195 participant pregnant women, 25(12.8%) with 95% CI (8.2%, 17.4%) were found to be colonized by GBS bacteria. None of the independent variables had statistically significant association with GBS anogenital colonization with p value <0.05. Conclusion: The results of this study showed prevalence of anogenital GBS colonization is significant which calls for routine screening near term and further epidemiologic studies in the future. Key words: Group B streptococcus(GBS), prevalence, anogenital colonization.