Objectives: To evaluate the use of real-time polymerase chain reaction (RT-PCR) and bacterial culture methods to detect group B streptococcus (GBS) in Chinese pregnant women in the third trimester; to separately assess the prevalence of rectal and vaginal GBS colonization ; and to determine the antimicrobial susceptibility pattern of the isolates. Methodology: Samples were collected from 505 women at 35 and 37 weeks gestation at the Peking Union Medical College Hospital. Bacterial culture and RT-PCR were performed. Antimicrobial susceptibility to commonly used antibiotics was also analyzed. Results: The overall GBS colonization rate was 7.5%. The colonization rate, sensitivity, and negative predictive value of the bacterial culture method were 2.8%, 36.8%, and 95.1%, respectively, and these values were 7.3%, 97.4%, and 99.8%, respectively, for PCR (p<0.001). The GBS colonization rate of the rectum (6.7%) was higher than that of the vagina (2.8%) (p=0.005). Antimicrobial susceptibility testing showed that 100% were sensitive to penicillin, cephalosporin and vancomycin. Conclusions: RT-PCR was found to be a rapid and sensitive test for the detection of GBS colonization in Chinese pregnant women. Rectal swabbing was also important for detecting GBS colonization. β-lactams are the first-line antibiotics used for the treatment of GBS.