OBJECTIVE Estimate the incidence, risk factors and clinical features of group B streptococcal (GBS) disease in Australian infants and compare bacterial genotypes between infant disease and maternal carriage. METHODS The Australian Paediatric Surveillance Unit conducted a study of invasive GBS disease in infants aged 0-90 days between July 2005 and June 2008. Clinical data were obtained by questionnaire. GBS isolates from affected infants and antenatal carriers were referred for genotyping. RESULTS One hundred fifty confirmed cases were reported: 88 early-onset (EOD; 0-6 days) and 62 late-onset disease (LOD). Based on review of selected laboratory records, they represented ∼1/3 of all cases. Estimated national EOD and LOD rates were 0.38 and 0.19/1000 live births, respectively. Admission to intensive care was common (44%), and 7% of infants died. One or more obstetric indications for intrapartum antibiotic prophylaxis (IAP) were identified in 51% of mothers; 53% of mothers were screened for GBS carriage, and screening was positive in 45%; only 25% of women with clinical or microbiological risk factors were given IAP (no significant differences between EOD and LOD groups). Distribution of GBS genotypes differed significantly between invasive and vaginal isolates: virulent serosubtype III-2/sequence type 17 was strongly associated with LOD but uncommon among EOD and vaginal isolates. CONCLUSIONS Estimated GBS disease rates remain relatively low despite poor predictive values of clinical and microbiological criteria for, and compliance with, IAP. Clinical and microbiological epidemiology of LOD differs significantly from that of EOD. Further reduction in infant morbidity and mortality from GBS disease will require new strategies.