The aim of this study was to evaluate the effect of increasing screening-to-labor interval on the performance of group B streptococcus (GBS) screening by late-pregnancy enriched culture compared with intrapartum real-time polymerase chain reaction (RT-PCR). Group B streptococcus colonization was determined in 2624 women with singleton pregnancies by culture at 35-37weeks of gestation and at the beginning of labor by culture and RT-PCR from recto-vaginal swab samples. Group B streptococcus colonization rates were 29.0% in late-pregnancy culture, 29.7% in intrapartum culture and 28.2% in intrapartum PCR. Intrapartum culture was used as a reference, the late-pregnancy culture had an overall sensitivity of 89.2% (95% CI 88.0%-90.4%) and specificity of 96.5% (95% CI 95.8%-97.2%), and intrapartum PCR had sensitivity of 91.5% (95% CI 90.4%-92.6%) and specificity of 98.5% (95% CI 98.0%-99.0%). However, up to 4weeks after screening, the sensitivity of late-pregnancy culture was equivalent to or higher than that of RT-PCR. The RT-PCR was invalid in 0.9% of the women. Between late-pregnancy screening and labor, GBS colonization changed from negative to positive in 3.2% and from positive to negative in 2.5% of the women. The late-pregnancy enriched culture and intrapartum RT-PCR have comparable sensitivities in the detection of GBS when culture screening is conducted no more than 4weeks before labor. Late-pregnancy culture sampling should be postponed to at least 37weeks of gestation.