This study identifies a 35-kbp genetic locus, named Locus of Enterocyte Effacement (LEE), in *Enteropathogenic Escherichia coli* (EPEC) that is responsible for the formation of attaching and effacing (AE) lesions on intestinal epithelial cells. The LEE locus is conserved among various enterobacterial pathogens, including EPEC, *Enterohemorrhagic E. coli* (EHEC), *RDEC-1*, *Hafnia alvei*, and *Citrobacter freundii* biotype 4280. The LEE locus contains multiple regions implicated in AE lesion formation, such as the *eaeA* and *eaeB* genes. The study also reveals that the LEE locus inserts at the same site on the *E. coli* chromosome as a virulence locus in uropathogenic E. coli, suggesting evolutionary relatedness. The findings suggest that the LEE locus may be a cluster of virulence genes that spread horizontally among unrelated bacteria, potentially through homologous recombination or site-specific recombination systems. The conservation of the LEE locus among diverse pathogens highlights its importance in the pathogenesis of AE lesions and provides insights into the mechanisms of pathogen evolution.This study identifies a 35-kbp genetic locus, named Locus of Enterocyte Effacement (LEE), in *Enteropathogenic Escherichia coli* (EPEC) that is responsible for the formation of attaching and effacing (AE) lesions on intestinal epithelial cells. The LEE locus is conserved among various enterobacterial pathogens, including EPEC, *Enterohemorrhagic E. coli* (EHEC), *RDEC-1*, *Hafnia alvei*, and *Citrobacter freundii* biotype 4280. The LEE locus contains multiple regions implicated in AE lesion formation, such as the *eaeA* and *eaeB* genes. The study also reveals that the LEE locus inserts at the same site on the *E. coli* chromosome as a virulence locus in uropathogenic E. coli, suggesting evolutionary relatedness. The findings suggest that the LEE locus may be a cluster of virulence genes that spread horizontally among unrelated bacteria, potentially through homologous recombination or site-specific recombination systems. The conservation of the LEE locus among diverse pathogens highlights its importance in the pathogenesis of AE lesions and provides insights into the mechanisms of pathogen evolution.