Molecular evidence is limited for the hypothesis that humans, dogs, and cats can become colonized and infected with similar virulent Escherichia coli strains. To further assess this possibility, archived E. coli O6 isolates (n = 130) from humans (n = 55), dogs (n = 59), and cats (n = 16), representing the three main H (flagellar) types within serogroup O6 (H1, H7, and H31), were analyzed, along with selected reference strains. Isolates underwent PCR-based phylotyping, multilocus sequence typing, PCR-based detection of 55 virulence-associated genes, and XbaI pulsed-field gel electrophoresis (PFGE) profiling. Three major sequence types (STs), which corresponded closely with H types, accounted for 99% of the 130 O6 isolates. Each ST included human, dog, and cat isolates; two included reference pyelonephritis isolates CFT073 (O6:K2:H1) and 536 (O6:K15:H31). Virulence genotypes overlapped considerably among host species, despite statistically significant differences between human and pet isolates. Several human and dog isolates from ST127 (O6:H31) exhibited identical virulence genotypes and highly similar PFGE profiles, consistent with cross-species exchange of specific E. coli clones. In conclusion, the close similarity in the genomic backbone and virulence genotype between certain human- and animal-source E. coli isolates within serogroup O6 supports the hypothesis of zoonotic potential.