Background & Objective:This study was designed to determine the prevalence of serotypes, virulence-associated genes, and antimicrobial resistance ofStreptococcus agalactiaein pregnant volunteers attending a major maternity hospital in Iran.Methods:The virulence determinants and antimicrobial resistance profiles of 270 Group B streptococcus (GBS) samples were assessed in the adult participants. The prevalence of GBS serotypes, virulence-associated genes, and antimicrobial resistance of the isolates was determined.Results:The GBS prevalence in the vaginal, rectal, and urinal carrier rates were 8.9%, 4.44%, and 4.44%, respectively, with no concomitant colonization. The serotypes Ia, Ib, and II were at a 1:2:1 ratio. The rectal isolates, harboringCylE,lmb, andbcagenes, were of serotype Ia, susceptible to vancomycin. The serotype Ib from urine samples carrying three distinct virulence genes was susceptible to Ampicillin. In comparison, the same serotype with two virulence genes ofCylEandlmbexhibited sensitivity to both Ampicillin and Ceftriaxone. The vaginal isolates belonged to serotype II with the CylE gene or serotype Ib withCylEandlmbgenes. These isolates harboring theCylEgene were resistant to Cefotaxime. The overall antibiotic susceptibility range was 12.5-56.25%.Conclusion:The findings broaden our understanding of the pathogenicity of the prevailing GBS colonization and predict different clinical outcomes.