Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates but is preventable if the mother is diagnosed before and treated at delivery. Using 200 vaginal-rectal swabs inoculated to enrichment (LIM) broths, we compared routine culture and 2 real-time polymerase chain reaction (PCR) assays for detection of GBS: the LightCycler (LC) Strep B analyte-specific reagents (ASRs) (Roche Diagnostics, Indianapolis, IN) and the BD GeneOhm StrepB (BD-StrepB) test (BD GeneOhm Sciences, San Diego, CA). Culture detected 26.5% GBS-positive specimens, whereas the LC Strep B ASR and BD-StrepB test identified 29.5% and 30.0% positive specimens, respectively. Because of the increased detection rate of 3.0% to 3.5% observed with PCR, a second GBS-specific amplicon was sequenced to confirm the presence of GBS that was not detected by culture. In our hands, the sensitivity/specificity of the LC Strep B ASR was 100%/95.9%, and the BD-StrepB test was 92.5%/92.5% using culture as the gold standard.