OBJECTIVE To determine the prevalence of group B Streptococcus (GBS) colonization in two anatomical sites in Mexican women of childbearing age, as well as the associated risk factors. METHODS A retrospective cohort analysis was made with two groups of women between 14 and 44 years old. We formed two groups of 72 women; one had a recent positive group B Streptococcus culture and the other, paired by age and culture date, had a negative culture of the same bacterium. All of them answered a validated questionnaire and two rectal swabs were obtained and cultured for isolation of group B Streptococcus. RESULTS Prevalence of GBS colonization was 18% for two anatomical sites (heavy colonization), and 37% for one anatomical site; the overall prevalence was 54.9%. Three or more sexual partners increased by 4-fold the risk of heavy GBS colonization (p < 0.01). In this study 49 (34%) women were pregnant, 26 (53%) of them were colonized by GBS and 7 (14.3%) were heavy colonized. CONCLUSIONS Prevalence of GBS during pregnancy in Mexican women was higher than the reported previously, even in developed countries. Risk of colonization increased by 4-fold with a history of three or more sexual partners.