Background This study analyzed the clinical characteristics and serotype distribution of Group B streptococcus (GBS) in infants with invasive bacterial infection over a 20-year period according to diagnoses, age of onset and time period. Methods GBS isolates obtained from infants with invasive GBS disease at Seoul National University Children’s Hospital (1994-2013), Seoul National University Bundang Hospital (2003-2013) and Chonbuk National University Hospital (2001-2013) were analyzed for capsular serotype by PCR and antimicrobial susceptibility by microbiology laboratory and E-test. Clinical records were reviewed retrospectively. Results Among 58 cases with invasive GBS disease, 14 (24.1%) were early onset disease (EOD, 0-6 days), 42 (72.4%) were late onset disease (LOD, 7-89 days) and 2 (3.4%) were late-late onset disease (LLOD, 90-180 days). The most common diagnosis among EOD was bacteremia (35.7%), followed by meningitis (28.6%), pneumonia (21.4%) and osteomyelitis or septic arthritis (14.3%). In LOD, meningitis (52.4%) was the most common diagnosis, followed by bacteremia (42.9%) and osteomyelitis or septic arthritis (4.8%). The predominant serotypes were III (43.1%), V (31%), Ia (13.8%) and Ib (10.3%).There was no difference in the serotype distribution between EOD and LOD. According to disease, serotype III was responsible for 50% of meningitis and serotype V accounted for 42.9% in bacteremia. No penicillin-resistant strains were detected, however 46.5% of the strains had resistance to erythromycin, and 53.4% showed clindamycin resistance. The case fatality rate was 6.9% with no difference between EOD and LOD. There were sequelae in 13 cases (22.4%) with 11 cases of LOD (26.2%) and 2 cases of EOD (14.3%). Conclusions Among infants with invasive GBS disease in Korea, a majority of the cases were LOD. Serotype III was the most common followed by V. Surveillance studies on invasive GBS disease along with molecular analysis of GBS should be continued.