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Background: Group B Streptococcus (GBS) is a known leading causative pathogen of neonatal infection. Efficient screening and identification of women colonized with GBS is important for the prevention of invasive neonatal infections. Methods: A total of 628 vaginal/rectal specimens were collected from pregnant women in Beijing, China. The chromogenic medium chromID Strepto B agar (STRB) was evaluated for its reliability in screening GBS from the vaginal/rectal swabs; results were compared to those of blood agar plates (BAP). Furthermore, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) was used to confirm the colonies suspected of being GBS on STRB. Results: STRB showed excellent performance for GBS detection and outperformed BAP due to its higher sensitivity. Furthermore, MALDI-TOF MS could reliably differentiate the putative GBS isolates on STRB. Conclusions: This study demonstrated that STRB combined with MALDI-TOF MS is a fast, sensitive, and accurate method for the identification of GBS-colonized pregnant women.