Aims: To determine the degree of relatedness between isolates of Escherichia coli O157:H7 of human, bovine, ovine and porcine origin. Methods and Results: Escherichia coli O157:H7 isolates were compared using (i) PFGE XbaI patterns, (ii) PCR profiles of virulence genes and (iii) the DNA sequences of genes reported to play a role in pathogenicity. The 77 E. coli O157:H7 isolates demonstrated 49 different PFGE patterns of which, eight were common to multiple isolates, and the remaining 41 were distinct. Isolates of different origin did not correlate, except for one cluster consisting of two human and two beef isolates. The majority of animal isolates had the same PCR profiles of virulence genes as those isolated from clinical patients. Single nucleotide polymorphisms (SNPs) were identified in the sequence of a 255-bp region of the vtx2 subunit A gene. Conclusions: Six SNPs were detected in the vtx2A gene, defining four different haplotypes. One nonsynonymous substitution encoded for an amino acid change from glutamic to aspartic acid. Significance and Impact of the Study: Results indicate that although E. coli O157:H7 isolates of differing origin were distinct by PFGE, the DNA sequences of the main virulence genes associated with human clinical illness were conserved.