Group B Streptococcus (GBS) infection has long been recognized as a frequent cause of morbidity and mortality in newborn infants. The purpose of this study was to determine the colonization rate with GBS and the antibiotic susceptibility profile in pregnant women attending Gynecological clinics in Egypt. One-hundred and fifty vaginal swabs were collected from pregnant women at 35-40 weeks of gestation. In comparison to culture, direct latex agglutination testing revealed 100% sensitivity and 93.75% specificity. Thirty-eight specimens (25.3%) were found to be positive for GBS. Each isolate was tested for susceptibility to penicillin G, ampicillin, cefotaxime, erythromycin, clindamycin and vancomycin. Erythromycin-resistant isolates were further classified by double-disk method. All isolates were susceptible to penicillin G, ampicillin and vancomycin. Resistance to cefotaxime was detected in three isolates (7.89%). Five isolates (13.15%) were resistant to erythromycin and nine isolates (23.68%) were resistant to clindamycin. Four (80%) isolates had constitutive macrolide-lincosamide-Streptogramin(B) resistance (cMLSB(B)) resistance and one (20%) isolate had inducible resistance (iMLS(B)) resistance. GBS colonization was found to be high in our region. Latex agglutination testing and Islam medium are reliable methods to detect GBS in late pregnancy; however, latex agglutination test is rapid and simpler. Penicillin G remains the first choice antibiotic for treatment of GBS infections.