Background:  Group B Streptococcus (GBS) transmitted from colonized pregnant women to the newborns is considered the leading cause of neonatal sepsis with high mortality rate, so screening pregnant women for GBS colonization is important for intrapartum antibiotic prophylaxis to prevent neonatal and maternal infections, this study aimed to detect the prevalence of GBS colonization in pregnant women and to assess the diagnostic value of GeneXpert PCR assay in screening. Methods: Rectovaginal swabs from 318 pregnant women with gestational age 35 or more weeks were tested by conventional culture as a standard screening method and by GeneXpert PCR assay as a rapid molecular method. Results: The prevalence of GBS colonization in pregnant women was 17% by standard culture method and 22% by the GeneXpert PCR assay with overall 98.2% sensitivity, 93.6% specificity, 94.3% diagnostic accuracy and total turnaround time less than 1 hour. Conclusion: The increase in prevalence of GBS colonization of pregnant women raises the concern about importance of the accurate and rapid molecular diagnostic techniques for effective prevention of GBS transmission.