Background: Group B Streptococcus (GBS) (Streptococcus agalactiae) is the leading cause of morbidity and mortality of newborn infants considered a leading factor causing septicemia after birth. The standard method for the diagnosis of GBS colonization is culture in a selective medium, but PCR has a high sensitivity and specificity. Objectives: The goal of this study was to estimate the colonization of GBS in rectum of neonates of high-risk mothers by culture and PCR method. Patients and Methods: Samples were taken from rectal mucosa of 154 neonatesof high-risk mothers for GBS by swabs. Samples were tested by standard culture using Todd Hewitt broth and blood agar and also by PCR using primers specific for cfb gene. Results: Of 154 neonates, Culture identified 17 (11%) neonates as colonized by GBS; and the PCR assay could identify 27 (17%) neonates with positive results for GBS. Mothers age range was 17-40 years (mean = 26.1 ± 5.1). Maternal age was significantly lower in PCR positive group (P = 0.038) and in culture positive group (P = 0.015). Using culture as the gold standard, sensitivity, NPV, specificity, and PPV of PCR were 100%, 100%, 92%, and 62%, respectively. The time required for PCR assay and culture were 2hours and 36hours, respectively. Conclusions: This study showed that the incidence of GBS in Iranian high-risk neonates is high, so we strongly recommend screening of high-risk neonates for detection of GBS.