The goal of this study was to determine the prevalence of fluoroquinolone resistance in Streptococcus agalactiae and the serotype distribution of this resistant bacterium. S. agalactiae strains collected from 221 asymptomatic pregnant women (35-37 weeks of gestation) and 838 patients with S. agalactiae infection in Korea, from 2006 to 2008, were tested for susceptibility to four fluoroquinolones. Rates of resistance of S. agalactiae to ciprofloxacin, levofloxacin, and moxifloxacin were 9.3 %, 9.5 %, and 0.8 %, respectively; greater than 94 % of S. agalactiae strains were resistant to norfloxacin. Resistance to ciprofloxacin and levofloxacin increased between 2006 and 2008. All strains were susceptible to penicillin. Resistance to ciprofloxacin and levofloxacin was higher in the clinical strains of S. agalactiae isolated from infections than in colonizing strains isolated from pregnant women. Mutations in the quinolone resistance-determining regions of gyrase and topoisomerase genes were detected in strains resistant to ciprofloxacin, levofloxacin, and moxifloxacin; no such mutations were found in strains resistant only to norfloxacin. There was a strong correlation between the minimum inhibitory concentrations and the presence of mutations in gyrase and topoisomerase genes. In conclusion, the prevalence of fluoroquinolone resistance was unexpectedly high. Strain serotypes were not associated with susceptibility to fluoroquinolones.