OBJECTIVE To analyse antimicrobial susceptibility and serotypes of group B streptococcus (GBS) bloodstream isolates from different patient groups. METHODS Susceptibility to penicillin, erythromycin and clindamycin was measured for 99 bloodstream GBS isolates collected between October 2000 and July 2005. Multiplex PCR-based reverse line blot (mPCR/RLB) assays were used to identify macrolide resistance genes and capsular serotype for each isolate. Clinical correlation was obtained from chart review. RESULTS Adult bacteraemia accounted for 84 of 99 (85%) isolates, and were usually associated with underlying diseases such as diabetes, malignancy and renal failure. Overall mortality was 10%. Known macrolide resistance genes [ermB (2), ermA/TR (3) and mefA/E (2)] were detected in seven of eight erythromycin resistance isolates. Four of these isolates expressed MLSB phenotype, two with constitutive (ermB) and two with inducible (ermA/TR) clindamycin resistance. Of four M phenotype isolates, two had mefA/E, one had ermA/TR and one had no detectable macrolide resistance genes. Serotype III was significantly more common in neonatal isolates; serotype V was more common among adult isolates and was associated with increased mortality. CONCLUSIONS mPCR/RLB is a rapid molecular method to identify GBS serotype and macrolide resistance genes. This is the first major study correlating these characteristics with demographic data for invasive isolates.