Background: Globally, about 10-30% of the pregnant women were colonized with Group B Streptococcus in the genitourinary tract and 60% of their neonates were acquiring the infection during labor and delivery process. Methods: A cross-sectional study was conducted among 329 pregnant women at 35th-37th gestational weeks from June 06-2022 to July 22-2022 at selected health institutions, in Hawassa, Ethiopia. Socio-demographic and clinical data were obtained by using a structured questionnaire. Group B Streptococcus was isolated from vagino-rectal swabs by using standard method recommended by Center of Disease Control and Prevention and an antibiotic susceptibility test was performed according to the Clinical and Laboratory Standards Institute guidelines. Data were analyzed by using SPSS version 25.0. Bivariate logistic regression was carried out and variables with p < 0.25 were selected for multivariate logistic regressions, p < 0.05 were accepted as statistically significant. Results: The prevalence of Group B Streptococcus colonization among pregnant women at 35th-37th gestational weeks was 48/326 (14.7%; 95%CI: 10.8-18.5). The isolates that were developed resistance to Penicillin (2.1%), Ampicillin (4.2%), Erythromycin (8.3%), and Clindamycin (12.5%). Vancomycin showed (100%) sensitivity. Maternal GBS colonization were significantly associated with rural dwellers (AOR = 2.29 (95%CI = 1.17-5.32), p = 0.032) and who had one time ANC visit (AOR = 2.49 (95% CI = 1.07-4.93), p = 0.018). Conclusion: There was a high frequency of GBS colonization, relatively low resistance to commonly used antibiotics, and significant association of maternal GBS colonization with rural dwellers and one time antenatal visit which suggests the screening of GBS colonization in pregnant women at 35th-37th weeks of gestation, testing their antimicrobial susceptibilities and encouraging antenatal fellow especially in rural dwellers in order to reduce maternal GBS colonization, provide antibiotic prophylaxis and minimize newborn infection and co-morbidity.
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