Background: Streptococcus agalactiae is the major cause of meningitides and septicemia in infants and also intrauterine infection and preterm labor in pregnant women. This study aimed to determine the prevalence of group B streptococci (GBS) colonization in rectovaginal region of 15 to 40 years, pregnant women. Methods: Recto-vaginal swab samples from 250 pregnant women referred to health centers in the city of Yazd, Iran, 2002, were taken. The specimens were cultured on sheep blood agar and the presence of GBS was identified with catalase-negative, hipporte hydrolysis, CAMP (Christie Atkins Munch-Petersen) test, and Gram staining. Results were confirmed by polymerase chain reaction (PCR) molecular methods for the amplification of cpsH capsular gene. Findings: Out of 250 samples, 49 (19.6%) were identified as Streptococcus agalactiae. All 49 samples were PCR positive with cpsH gene fragment with a 550 bp fragment in length that were assessed with 1% gel agarose electrophoresis. The most prevalence was identified among women with 20-25 years old and the least one among 15-20. Conclusion: Statistics of GBS prevalence varies in different regions which could be due to various factors such as age, sex, culture method and the inherent differences in study populations and increased use of antibiotics in some populations. Due to high prevalence of GBS in Yazd, Iran, appropriate screen/control programs would be necessary to prevent the damages in infants and mothers. Keywords: Streptococcus agalactiae, Group B streptococcus, Rectovaginal, Polymerase chain reaction (PCR)