Objective: To explore factors associated with a high vaginal GBS load during labor considering (1) the recto-vaginal GBS load at 35–37 weeks’ gestation determined by culture and (2) the vaginal GBS colonization determined by a polymerase chain reaction (PCR) assay during labor. Methods: From an unselected cohort of 902 pregnant women, we obtained (1) recto-vaginal swabs for culture of GBS at 35–37 weeks’ gestation (GBS rectovag-36), (2) vaginal swabs for GBS PCR detection at labor (PCR vag-labor), and (3) vaginal swabs for culture of GBS at labor (GBS vag-labor). The GBS load was classified semi quantitatively according to a culture protocol without prior broth enrichment of the swab samples: none (0), few (+), some (++), or many (+++) GBS colonies. Results: Among 902 unselected pregnant women, 859 (95%) had a vaginal swab culture taken at labor, which was classified semi quantitatively. High load GBS vag-labor (+++) were found in 31 participants. GBS rectovag-36 showed a sensitivity of 90% (28/31) and a PPV of 23% (28/121), whereas PCR vag-labor had a sensitivity of 98% (30/31, non-significant difference) and a PPV of 42% (30/71, p <.01). PCR at labor had a lower sensitivity (78%) for detection of vaginal colonization with GBS at labor (any load) compared to recto/vaginal colonization with GBS at 36 weeks (92%). Vaginal colonization with GBS at 36 weeks seemed to have a lower sensitivity for detecting GBS in vagina at labor for high load (48%) and for any load (39%). Conclusion: PCR at labor has higher detection rate (non-significant) and PPV in identification of laboring women with a high load of vaginal GBS compared with recto-vaginal culture at 36 weeks’ gestation.