The Group B Streptococcus (Streptococcus agalactiae) (GBS) is one of the natural flora bacteria in the female reproductive system. In the recent years, GBS has become the major bacterial infections throw the perinatal period causing many troubles. This study was undertaken to determine the prevalence and antibiotic genes of GBS colonization in pregnant women of obstetrics hospital in Taif, Saudi Arabia. Fourteen Streptococcus agalactiae isolates obtained after screening about 134 swabs samples from genitourinary tract specimens of women patients from obstetrics hospital in Taif governorate, Saudi Arabia. These isolates were studied for antibiotic resistance and virulence genes. All obtained isolates were identified as Streptococcus agalactiae by the 16S rDNA gene sequence. These strains were found by Disc diffusion method sensitive against Meropenem, Cefotaxime, Cefepime, Amoxicillin, Penicillin G, Daptomycin, Chloramphenicol, Linezolid and Levofloxacin. The highest resistance was for Tetracyclin (85.7%) whereas the lowest resistance was found for Vancomycin (21.4%). Resistance against Erythromycin and Clindamycin was 71.5%, and 28.5% respectively. PCR based detection revealed 50% of isolates were carrying the tetT genes, while 92.8% of isolates were carrying tetO and tetM genes associated with Tetracycline resistance. All isolates were harboring genes that associated with Erythromycin resistance like ErmB1, ErmB2, and Erm(A|TR) genes, but only 28.5% of isolates were carrying ErmTR gene. Molecular detection of virulence associated genes revealed that out of fourteen isolates of S. agalactiae, only one isolate was carrying the LinB gene, while all isolates were positive for MreA and VanA genes respectively. We can conclude that the GBS isolates were found sensitive to many antibiotic while most isolates were resistance to Tetracycline due to the existence of tetO and tetM genes. Resistance against Erythromycin and Clindamycin was 71.5%, and 28.5% respectively. The two erythromycin resistance genes (ErmB and ErmTR) were found in all isolates, while, the third erythromycin resistance gene Erm(A|TR) was found only in 25.8% of the isolates.