Group B Streptococcus (GBS) is one of the most important causative agents of neonatal sepsis. As administration of prophylactic antibiotics during labor can prevent GBS infection, routine screening for this bacterium in prenatal care before the onset of labor is recommended. However, many women present in labor without having undergone such testing during antenatal care, and the turnaround time of detection methods is insufficient for results to be obtained before delivery. Vaginal and anorectal specimens were collected from 270 pregnant women. Each sample was tested by Xpert GBS, qPCR, and culture for GBS detection. The overall prevalence of maternal GBS colonization was 30.7% according to Xpert GBS, 51.1% according to qPCR, and 14.3% according to cultures. Considering the qPCR method as the reference, the Xpert GBS had a sensitivity of 53% and specificity of 93%. Positive Xpert GBS results were correlated to marital status (married or cohabitating) and with prematurity as a cause of neonatal hospitalization. Positive cultures were related with ischemic-hypoxic encephalopathy requiring therapeutic hypothermia. Combined enrichment/qPCR and the Xpert GBS rapid test found a high prevalence of GBS colonization. The Xpert GBS technique gives faster results and could be useful for evaluating mothers who present without antenatal GBS screening results and are at risk of preterm labor, thus allowing institution of prophylactic antibiotic therapy.