Background: Group B Streptococcus (GBS) comprises the normal flora of the female urogenital tract and can be transferred to neonates during delivery, causing invasive diseases.This study was performed to investigate the colonization rate, antibiotic susceptibility, and serotype of GBS among Saudi pregnant women. Materials and Methods: In this cross-sectional study, vagino-rectal swabs from 400 pregnant women were collected over a period of one year. Identification of GBS isolates and determination of their antibiotic susceptibility were performed using the Microscan Walk Away system. The isolates were then typed using both latex agglutination and capsular genebased multiplex polymerase chain reaction assays. Results: Sixty (15.0%) subjects were colonized by GBS, with serotype Ia as the dominant type (30.0%) followed by serotype III and V (25.0%, each). Only 43 (71.7%) isolates were typed by latex agglutination, whereas the remaining isolates were not typable or were non-specifically typed as compared to the genotyping assay, which revealed the specific type of each GBS isolate. The highest resistance rates were observed for erythromycin and clindamycin (16.7%, each), which were mainly restricted to the prevalent serotypes. Conclusion This study is the first to report the distribution of GBS serotypes based on molecular genotyping in Saudi Arabia. GBS colonization was evident among pregnant women, and resistance to erythromycin and clindamycin was predominant among serotypes Ia, III, and V. Molecular genotyping using capsular gene-based multiplex PCR provided reliable typing of the investigated GBS isolates in terms of sensitivity and specificity as compared to conventional serotyping using latex agglutination.