This study investigated the prevalence of recto-vaginal Group B Streptococcus (GBS) colonization, serotype distribution, and antimicrobial susceptibility patterns among pregnant women in Dongguan, China. Recto-vaginal swabs were collected from pregnant women at gestational age 35-37 weeks between January 1st 2009 and December 31st 2014. Isolates were serotyped by latex-agglutination and were tested against seven antimicrobials by disk diffusion. Of 7,726 pregnant women who completed GBS testing, 636 (8.2%) were GBS carriers. Of 153 GBS isolates available for typing, 6 serotypes (Ia, Ib, III, V, VI and VIII) were identified with type III being predominant, while 9 (5.9%) were non-typable isolates. All isolates were sensitive to penicillin, ceftriaxone, linezolid and vancomycin, whereas 52.4% were resistant to clindamycin, 25.9% were resistant to levofloxacin and 64.9% were resistant to erythromycin. This study showed the recto-vaginal colonization prevalence of GBS in Dongguan is significant. Due to 100% susceptibility to penicillin of all GBS samples, penicillin remains the first recommendation for treatment and prevention against GBS infection. Susceptibility testing should be performed for women allergic to penicillin in order to choose the most appropriate antibacterial agents for treatment and prevention of vertical transmission to neonates. In addition, we suggest establishing standard processes for GBS culture and identification in China as early as possible.