Background & Objectives: One of the important infectious factors in pregnant mothers and newborns is Group B Streptococcus (GBS). There is no perfect report about prevalence of GBS in Iran and in the case of preterm rupture of amniotic membrane or preterm labor all patients are treated by antibiotics without culture so this has led to adverse taking antibiotics and drug resistance. The present study is intended to determine the frequency of Colonization of GBS in the pregnant mother (35-37 weeks), referred to medical centers of Shahed University.Methods: Overall, 210 pregnant women (35-37 weeks), referred to medical center of Shahed University, Tehran, Iran were selected as sample group and after filling out the questionnaires about demographic data and midwifery status and the related information of post- partum, the rectovaginal culture was done for them.Results: Among 210 samples, 7 (3.3%) included positive culture in terms of GBS Colonization while all these cases were sensitive to penicillin, ampicillin, cephalothin, nitrofurantoin, and all of the samples were resistant against tetracycline and contrimoxazole. There was no relationship among age, job, education, number of pregnancy, blood pressure background, diabetes and preterm childbirth with positive culture. Similarly, because of limited number of positive cases it was not possible to examine the relationship among GBS Colonization and infection in mother and newborn.Conclusion: There was a low frequency in GBS Colonization in the studied hospitals and the study inside the country also confirms this finding.