Background: Streptococcus agalactiae, or Lancefield group B Streptococcus (GBS), is carried by women of childbearing age at variable frequencies. It is responsible for high neonatal infection rates. Objective: Was to detect the frequency of Streptococcus agalactiae carriage among pregnant females attending Benha University Hospital, identify the serotype of the isolated strains and to study their antibiotic susceptibility profile. Methodology: Two hundred and fifty pregnant females between 35 and 37 weeks gestation were enrolled in this study. Rectovaginal swabs were collected. Identification, serotyping, antimicrobial susceptibility, erythromycin resistance phenotyping by double disc diffusion test were performed. Results: GBS colonization was detected in 28% of investigated subjects, with serotype V was found in 37.1% of them. All isolates were sensitive to benzylpenicillin, ampicllin, cephotaxim and vancomycin. Resistance to erythromycin and clindamycin was (42.8 and 17.2%) respectively. Inducible and constitutive resistance were 12.9 and 17.1 %. M phenotype was detected in 4.3% of GBS strains. Conclusion: the results of our study would be useful for implementing prenatal GBS screening and proper choice of antibiotic for intrapartum prophylaxis