Over the last decade, CTX-M enzymes have become the most prevalent extended-spectrum beta-lactamases (ESBLs) worldwide, mostly in Escherichia coli, causing a major health problem. An epidemiological relationship has been established between a rare genotype of E. coli, the D(2) genotype, and the presence of CTX-M genes. We investigated this striking association by exploring the genetic backgrounds of 18 D(2) genotype CTX-M-producing strains and of the plasmids encoding CTX-M enzymes. The 18 strains had different genetic backgrounds, as assessed by multilocus sequence and O typing, and were associated with various plasmids bearing diverse CTX-M genes. The region encompassing the genetic marker of the D(2) genotype (TSPE4.C2) was not correlated with the presence of CTX-M genes. CTX-M-producing D(2) strains had far fewer virulence factors than a control group of 8 non-ESBL-producing D(2) strains, and an inverse relationship was found between the number of co-resistances associated with the CTX-M gene and the number of virulence factors found in the strain. These findings provide evidence for multiple acquisitions of plasmids carrying CTX-M genes in different D(2) genotype strains. They strongly suggest that convergent evolution has occurred, and indicate that there has been selection for the association of a specific genetic background of the strain and the CTX-M gene. This fine-tuning of the relationship between the D(2) genotype and CTX-M genes presumably increases the fitness of the strain, indicating a role for the host cell in the acquisition and dissemination of CTX-M genes.