Escherichia coli is a common bacterial species in the gastrointestinal tracts of warm-blooded animals and humans. Pathogenicity and antimicrobial resistance in E. coli may emerge via host switching from animal reservoirs. Despite its potential clinical importance, knowledge of the population structure of commensal E. coli within wild hosts and the epidemiological links between E. coli in nonhuman hosts and E. coli in humans is still scarce. In this study, we analyzed the whole-genome sequencing data of a collection of 119 commensal E. coli strains recovered from the guts of 55 mammal and bird species in Mexico and Venezuela in the 1990s. We observed low concordance between the population structures of E. coli isolates colonizing wild animals and the phylogeny, taxonomy, and ecological and physiological attributes of the host species, with distantly related E. coli strains often colonizing the same or similar host species and distantly related host species often hosting closely related E. coli strains. We found no evidence for recent transmission of E. coli genomes from wild animals to either domesticated animals or humans. However, multiple livestockand human-related virulence factor genes were present in E. coli of wild animals, including virulence factors characteristic of Shiga toxin-producing E. coli (STEC) and atypical enteropathogenic E. coli (aEPEC), where several isolates from wild hosts harbored the locus of enterocyte effacement (LEE) pathogenicity island. Moreover, E. coli isolates from wild animal hosts often harbored known antibiotic resistance determinants, including those against ciprofloxacin, aminoglycosides, tetracyclines, and beta-lactams, with some determinants present in multiple, distantly related E. coli lineages colonizing very different host animals. We conclude that genome pools of E. coli colonizing the guts of wild animals and humans share virulence and antibiotic resistance genes, underscoring the idea that wild animals could serve as reservoirs for E. coli pathogenicity in human and livestock infections. IMPORTANCE Escherichia coli is a clinically important bacterial species implicated in humanand livestock-associated infections worldwide. The bacterium is known to reside in the guts of humans, livestock, and wild animals. Although wild animals are recognized as potential reservoirs for pathogenic E. coli strains, the knowledge of the population structure of E. coli in wild hosts is still scarce. In this study, we used fine resolution of whole-genome sequencing to provide novel insights into the evolution of E. coli genomes from a small yet diverse collection of strains recovered within a broad range of wild animal species (including mammals and birds), the coevolution of E. coli strains with their hosts, and the genetics of pathogenicity of E. coli strains in wild hosts in Mexico. Our results provide evidence for the clinical importance of wild animals as reservoirs for pathogenic strains and highlight the need to include nonhuman hosts in the surveillance programs for E. coli infections.Peer reviewe