Background: Streptococcus agalactiae or group B streptococcus (GBS) is a prominent cause of severe neonatal infections. GBS is a part of the intestinal and vaginal normal flora. Maternal colonization is recognized as the main path of GBS transmission. GBS is a pathobiont that changes from a non-symptomatic mucosal carriage state to a significant bacterial pathogen, causing major infections. Objectives: This study aimed to investigate the concomitant presence of major colonization genes, including ftsA, ftsB, lmb, and sfbA, and to determine the genetic relatedness of clinical GBS isolates. Methods: The GBS isolates were obtained from urinary and placental samples of pregnant women with a urinary tract infection, who were admitted to a hospital in Tehran, Iran. The presence of some major colonization factors was investigated via multiplex PCRassay. Genotyping of the isolates was performed using the BOX-PCR fingerprint technique with a BOX-A1R primer. Next, the data were analyzed using the UPGMA method and the coefficient of Jaccard in NTSYS software. Results: A total of 60 GBS isolates were examined in this study. The concomitant presence of target colonization genes was observed in all isolates. The BOX-PCR discriminated GBS isolates into sixdifferent genetic clusters at a 60% cutoff point. The majority of isolates (80%) from both clinical samples were clustered into genotypes 2, 6, and 4, while the rest (20%) were distributed equally into three different genotypes. Conclusions: Determining the colonization associated genes and genetic polymorphism in a different geographical area provides the epidemiological basis for the prevention of GBS infections in pregnant women and infants.