Abstract: Maternal recto-vaginal colonization of group B streptococcus in pregnant women visiting a hospital in Mashhad-Iran was studied. Recto-vaginal samples obtained from 201 pregnant women were carefully observed by direct Gram stain. Also, the samples were inoculated to Todd-Hewitt broth containing supplements. After re-inoculation onto sheep blood agar, group B streptococci were identified by colony morphology, beta-hemolysis, differential biochemical tests and agglutination test as confirmation identification. 25 (12.44%) of 201 pregnant women were GBS carriers; 24 (11.94%) and 22 (10.95%) positive for rectal and vaginal swabs, respectively. There were 2 (28.57%) carriers among 7 diabetic studied pregnant women. Gram positive bacilli suspected to lactobacilli in direct Gram stain of GBS carriers were observed less than non carriers, therefore replacing vaginal normal flora using lactobacilli as probiotic is recommended to prevent GBS infections in mothers and their neonates.