Escherichia coli is a leading cause of urinary tract infections. One of the most common antibiotic classes used to treat such infections is the ߭lactams, including cephalosporins. Resistance to the third-generation cephalosporins can be caused by production of extended-spectrum ߭lactamases (ESBLs) or plasmid-mediated AmpC ߭lactamases. The most commonly reported AmpC ߭lactamase in E. coli is CMY-2. Plasmid-mediated CMY-2 has been frequently reported in E. coli and Salmonella sp. from food-producing animals. This study aimed to elucidate the molecular characteristics of clinical E. coli isolates carrying plasmids encoding CMY-2. A total of 67 CMY-2-producing E. coli were characterised by clonal analysis and phylogenetic typing. Characterisation of the plasmids carrying blaCMY-2 included replicon typing, plasmid profiling, plasmid transferability and sequencing of the blaCMY-2 genetic environment. As a result, E. coli producing CMY-2 was found to be highly polyclonal. The majority of CMY-2-producing E. coli belonged to phylogenetic group D. IncI1 plasmids were predominant among those carrying blaCMY-2 (96%). Restriction analysis revealed a single IncI1 plasmid carrying blaCMY-2 to be predominant and present in different clones of E. coli. IS1294-ISEcp1 complex or ISEcp1 that was truncated by IS1294 was the predominant insertion sequence upstream of blaCMY-2. The homogeneous genetic environment of blaCMY-2 observed among different strains of E. coli strongly suggests horizontal transfer of this IncI1, blaCMY-2-carrying plasmid. In summary, horizontal plasmid transfer plays a major role in the spread of blaCMY-2 in E. coli.