Multidrug-resistant (MDR) Streptococcus pneumoniae (i.e. resistant to three different antimicrobial classes) is a global concern. The molecular epidemiology of MDR S. pneumoniae has not been characterised in Canadian paediatric isolates. Paediatric MDR S. pneumoniae were obtained from a national surveillance study. Susceptibility testing was performed by the methods of the Clinical and Laboratory Standards Institute. Phenotypic and genotypic relatedness were assessed by serotyping and pulsed-field gel electrophoresis (PFGE). Penicillin resistance was assessed with polymerase chain reaction (PCR) followed by DNA sequencing of penicillin-binding proteins (PBPs) 1A, 2B and 2X. Macrolide resistance was assessed by PCR-based detection of mef(E) and erm(B). PCR and sequencing of the dihydrofolate reductase (DHFR) gene was performed to assess resistance to trimethoprim/sulphamethoxazole (T/S). Seventy (98.6%) of 71 MDR paediatric isolates were concomitantly resistant to penicillin, erythromycin and T/S. Resistance genes mef(E) (66.2%) or erm(B) (22.5%) or both mef(E) and erm(B) (8.5%) were associated with macrolide resistance, and the prevalence of erm(B) increased significantly (P=0.0001) over time. Penicillin resistance was associated with amino acid substitutions in PBPs 1A, 2B and 2X. Resistance to T/S was associated with amino acid substitutions in the DHFR gene; in particular, Ile100-->Leu was detected in all isolates analysed. PFGE revealed three clusters of isolates that were genetically related and associated with specific serotypes (Taiwan(19F), Spain(23F), Spain(14) and France(9V)), suggesting clonal expansion as the primary means of paediatric MDR S. pneumoniae dissemination in Canada. The heptavalent pneumococcal vaccine Prevnar, currently approved in Canada for use in children < or =2 years of age, provided excellent coverage (90.2%) of paediatric MDR S. pneumoniae. In conclusion, paediatric MDR S. pneumoniae simultaneously resistant to penicillin, erythromycin and T/S are genetically similar and disseminating across Canada. Prevnar provides excellent coverage of paediatric MDR S. pneumoniae.