The genetic determinants involved in reduced susceptibility to third-generation cephalosporins and aztreonam were identified in ten canine Enterobacter isolates associated with opportunistic infections in three veterinary hospitals in Brisbane, Australia. All isolates were evaluated by a combination of phenotypic (broth microdilution and disc susceptibility, modified disc diffusion and IEF) and genotypic (PFGE, plasmid analysis, Southern blot hybridization, bacterial conjugation, PCR and sequencing) methods to investigate genetic relatedness and to identify plasmid-mediated resistance genes, in particular beta-lactamase genes responsible for extended-spectrum cephalosporin resistance. The ten canine isolates were genotypically diverse based on PFGE and belonged to either Enterobacter cloacae or Enterobacter hormaechei on the basis of 16S rRNA gene sequence analysis. Plasmid profiles were also diverse. Nine isolates contained a transmissible blaSHV-12-carrying plasmid (approximately 140 kb) that also conferred resistance to chloramphenicol, gentamicin, spectinomycin, tetracycline, trimethoprim and sulfonamides. In all plasmid-mediated extended-spectrum beta-lactamase (ESBL)-producing isolates including transconjugants, blaSHV-12 was shown to reside in a approximately 6.5 kb plasmid fragment. The remaining isolate that was not an ESBL producer possessed an AmpC beta-lactamase gene (blaCMY-2) on a approximately 93 kb transmissible plasmid. This plasmid did not contain any other antimicrobial resistance genes. Additional plasmid-mediated beta-lactamases identified in some isolates included bla(TEM) and blaOXA-10. This is the first report of canine Enterobacter isolates containing transmissible plasmid-mediated blaSHV-12 and blaCMY-2 resistance genes. Therefore, Enterobacter isolated from opportunistic infections in dogs may be an important reservoir of plasmid-mediated resistance genes, which could potentially be spread to other members of the Enterobacteriaceae.