We studied the molecular mechanisms of resistance and mobility of 18 multidrug-resistant CTX-M-producing Escherichia coli isolates isolated from patients in Canadian intensive care units. Fluoroquinolone-resistant isolates (83.3%) had mutations in gyrA and parC. Plasmid-mediated quinolone resistance genes qnr (A, B, and S), qepA, and aac(6')-Ib-cr were detected in 0%, 5.6%, and 44.4%, respectively. Sulfamethoxazole/trimethoprim-resistant isolates (61.1%) carried a dfr gene, and 10 (90.9%) of the 11 carried 1 or more sul genes. Gentamicin-resistant isolates (27.8%) carried the aac(3')-II gene, and doxycycline-resistant isolates (33.3%) carried 1 or more tet efflux genes. Both genetically related and unrelated groups of E. coli harboring extended-spectrum beta-lactamases were observed. The bla(CTX-M) genes were primarily located on diverse IncF plasmids of multiple replicon types downstream of the ISEcp1 element. The spread of the bla(CTX-M) genes among E. coli in Canada occurs through a diversity of different mechanisms and does not correspond to a single CTX-M determinant, or a single clone, or a single plasmid but rather through the combination of clonal spread of virulent strains and acquisition of diverse CTX-M-bearing plasmids. We report the 1st qepA-producing E. coli in North America.