Background: Group B streptococcus is one of the most common causes of severe neonatal infections. Aim: To detect the prevalence of group B. Streptococcus and their antimicrobial susceptibility and to assess the role of PCR as a rapid method of its detection. Place and Duration of the Study: A cross sectional and prospective cohort study was carried out from September 2019 to February 2020 in Gynaecology and Obstetrics OPD and inpatient units in Al- Quwayiyah General hospital, Riyadh, Saudi Arabia Methodology: Paired rectal/vaginal specimens were collected from 540 pregnant females with gestational age 35 or more, Each swab was inoculated into selective medium, Todd Hewitt, One swab is streaked onto blood agar plates incubated in 5% CO2 for 24h at 36ºC. β-hemolytic colonies growth is identified by Gram’s stain, colony morphology and CAMP test. The confirmation and antimicrobial susceptibility were done by Vitek II machine, The second swab was used for PCR to identify atr gene. Results: Out of 540 pregnant women 87 (16.1%) were colonized with GBS isolates. The positive GBS women aged 25 (22-34) and negative GBS women aged 23 (24-35) with no statistical difference. Patients aged more than 35 years old has the higher rate of positive GBS, 46.2%. No significant association detected between GBS and gestational age at delivery, antenatal visits, BMI and gravidity. The GBS strains isolated from pregnant women was 100% susceptible to linezolid and vancomycin followed by ampicillin (93.1%) and tobramicin (83.9%) then gentamicin (81.6%) and levofloxacin(78.2%) and showed least antibiotic susceptibility to erythromycin (26.4%). The PCR was positive in 145 (26.9%). Using culture as gold method, PCR sensitivity was 100% (95% CI: 91.62-100), while specificity was 83.2% (95% CI: 82.61-91.02). Negative and positive predictive values were 100% and 61% respectively. Kappa between the two methods was 0.71, which indicate major agreement. Conclusion: The GBS prevalence among the pregnant females in Al Quwayiyah General Hospital was 16.1%. Detection of GBS using new PCR technique was found to have high sensitivity and faster results, allowing efficient management of GBS and reduction in newborn morbidity and mortality however the cost is high for some laboratories. Further studies should be assessed to be both low cost and accurate rapid screening.