Background:The aim of this study was to determine the prevalence of group B Streptococcus (GBS) colonization and to evaluate the antimicrobial resistance profile in women in the third trimester of pregnancy. Materials and Methods: A total of 310 pregnant women, referred in weeks 35 to 37 of gestation, were screened for GBS colonization during a 10-month period. Samples were collected from the vagina and the rectum. Results: The colonization rate was 10.6 % and 22 women (66.7%) had both positive vaginal and rectal cultures. Rates of GBS colonization were significantly lower in patients aged 24 years or older and in those with a third or later pregnancy. None of the isolates were resistant to penicillin and ampicillin, whereas 21.2% and 9.1% showed resistance to erythromycin and clindamycin, respectively. Conclusion: Screening and antimicrobial susceptibility testing of GBS during pregnancy are important to guide appropiate therapy.