Group B Streptococcus (GBS) is the leading cause of early-onset neonatal sepsis. This prospective cohort study aimed to determine the sensitivity and specificity of intrapartum PCR in detecting GBS colonization in comparison to antenatal culture. Patients who presented in labor or for induction of labor, ≥ 37 weeks’ gestation, with antenatal GBS swab cultures collected during routine screening within 5 weeks of delivery were recruited. A double-vaginal intrapartum swab was taken for GBS PCR assay and culture. The performance of antenatal culture and intrapartum PCR were evaluated using the corresponding intrapartum culture as the reference standard. Among 170 included participants, antenatal culture and intrapartum PCR have comparable sensitivities of 92.1% (95% confidence interval, 78.6 – 98.3) and 89.2% (74.6 – 97.0) (p=0.655) respectively. However, intrapartum PCR has a higher specificity of 79.6% (71.7 – 86.1) compared to 61.4% (52.5 – 69.7) (p<0.001) in antenatal culture. The false positive rates for intrapartum PCR (19.2%) were lower than those for antenatal culture (38.6%). Our study suggests that intrapartum PCR is a rapid and effective test to detect GBS colonization, with comparable sensitivity and superior specificity to antenatal swab culture. This strategy may reduce unnecessary intrapartum antibiotics exposure among women who are GBS negative during labor.