Objectives: To determine clonality and identify plasmid-mediated resistance genes in 11 multidrug-resistant Escherichia coli (MDREC) isolates associated with opportunistic infections in hospitalized dogs in Australia. Methods: Phenotypic (MIC determinations, modified double-disc diffusion and isoelectric focusing) and genotypic methods (PFGE, plasmid analysis, PCR, sequencing, Southern hybridization, bacterial conjugation and transformation) were used to characterize, investigate the genetic relatedness of, and identify selected plasmid-mediated antimicrobial resistance genes, in the canine MDREC. Results: Canine MDRECs were divided into two clonal groups (CG 1 and 2) with distinct restriction endonuclease digestion and plasmid profiles. All isolates possessed blaCMY-7 on an ~93 kb plasmid. In CG 1 isolates, blaTEM, catA1 and class 1 integron-associated dfrA17-aadA5 genes were located on an ~170 kb plasmid. In CG 2 isolates, a second ~93 kb plasmid contained blaTEM and unidentified class 1 integron genes, although a single CG 2 strain carried dfrA5. Antimicrobial susceptibility profiling of E. coli K12 transformed with CG 2 large plasmids confirmed that the blaCMY-7-carrying plasmid did not carry any other antimicrobial resistance genes, whereas the blaTEM/class 1 integron-carrying plasmid carried genes conferring resistance to tetracycline and streptomycin also. Conclusions: This is the first report on the detection of plasmid-mediated blaCMY-7 in animal isolates in Australia. MDREC isolated from extraintestinal infections in dogs may be an important reservoir of plasmid-mediated resistance genes.