Maternal vaginal/rectal colonization of group B streptococcus (GBS) is a main risk for neonatal invasive infection. Efficient determination of GBS colonization in pregnant women is crucial. This study aimed to investigate the prevalence of GBS carriage and evaluate the diagnostic performance of six methodologies for GBS screening conducted in China, including blood agar plate, liquid chromogenic medium, and loop-mediated isothermal amplification (LAMP) without pre-enrichment, chromogenic agar plate with pre-enrichment, and GBS antigen detection without and with pre-enrichment in comparison with the standard reference method (Lim broth-enriched subculture with plating on 5% sheep blood agar). Vaginal/rectal swabs were collected from 1,281 pregnant women at 35–37 weeks of gestation. Of them, 309 were taken in triplicate, one for Lim broth-enriched subculture, one for blood agar plate, and the third for GBS antigen detection (Reagent W); 177 were acquired in duplicate, one for Lim broth-enriched subculture and the other for GBS antigen detection (Reagent H); 502 were obtained in duplicate, one for Lim broth-enriched subculture and the other for liquid chromogenic medium; 158 were collected in duplicate, one for Lim broth-enriched subculture and the other for LAMP; and 135 were inoculated in Lim broth-enriched for GBS antigen detection (Reagent W) and subculture with chromogenic agar plate and 5% blood agar plate. The overall prevalence of GBS carriage was 10.1% (130/1,281, 95% CI: 8.5–12.1%) according to the standard reference method. Compared with the standard reference method, the LAMP had excellent performance of sensitivity (100%, 95%CI: 83.4–100%), specificity (94%, 95%CI: 88.1–97.1%), and Yoden index (0.940); as well as the blood agar plate with sensitivity (81.5%, 95%CI: 61.3–93.0%), specificity (100%, 95%CI: 98.3–100.0%), and Yoden index (0.815). The other four methods were not sufficient to reach the threshold in terms of sensitivity or specificity compared to the standard reference method. Furthermore, for LAMP, results can be obtained within 0.5–1 h, while for blood agar plate, which needed 24–48 h, and further identification was required. Our data suggested that the performance of LAMP was highly comparable to the standard Lim broth-enriched subculture and LAMP is considered as an alternative for fast and accurate GBS screening.