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Abstract Objectives: to assess the prevalence and epidemiological factors associated with group B Streptococcus (GBS) colonization in pregnant women in Porto Velho City, Rondônia. Methods: GBS was identified and isolated by genotypic and microbiological methods from rectovaginal samples of pregnant women between 35 and 37 weeks of gestation. Epidemiological data were collected using questionnaires and their correlation with colonization was assessed. The antimicrobial susceptibility profile was determined by disk diffusion method. Results: a total of 22.5% (102/453) pregnant women were colonized with GBS. A higher level of colonization was observed at the vaginal tract (17.6%), compared to the rectal area. We did not find any sociodemographic or obstetric factors associated with an increased risk of GBS colonization. All strains were susceptible to antibiotics penicillin, ampicillin, cefazolin, and ceftriaxone. In contrast, the rates of resistance to tetracycline (74.1%), erythromycin (14.1%), and clindamycin (3.5%) were observed. Conclusion: the prevalence of GBS as well as the absence of predictors of colonization demonstrated the need for universal screening for GBS in all pregnant women in the region. In addition, we showed that the first-line antibiotics recommended for prophylaxis are still good options for the prevention of neonatal GBS disease in the region.