Enterobacter sakazakii, designated a unique species in 1980, has been implicated as the causative organism in a rare but severe form of neonatal meningitis. Dried infant formula milk has been identified as a potential source of the organism. E. sakazakii isolates from dried infant formula available in Canada and clinical isolates obtained from Canadian hospital culture collections were characterised by phenotypic (biotype and antibiograms) and genotypic (ribotyping, random amplification of polymorphic DNA and pulsed-field gel electrophoresis) methods. Three biotypes and four antibiogram patterns were observed in the 18 isolates examined. Ribotyping with the Dupont Riboprinter microbial identification system divided the 18 isolates into 10 ribotypes. Three isolates from the same hospital had indistinguishable ribotyping patterns although each was isolated in a different year, as did three food isolates from one company. Pulsed-field gel electrophoresis (PFGE) and random amplification of polymorphic DNA (RAPD) profiles indicated minor differences between the isolates that were indistinguishable by ribotyping. PFGE (with the restriction endonucleases Xba1 and Spe1) and RAPD gave discrete patterns that enabled easy comparison of E. sakazakii isolates, with a high degree of discrimination. The discriminatory index showed RAPD and PFGE were shown to be the most discriminatory typing schemes for E. sakazakii, followed by ribotyping, biotyping and antibiograms.