Shiga toxin-producing Escherichia coli (STEC) strains cause a wide spectrum of diseases in humans. In this study, we tested 206 STEC strains isolated from patients for potential virulence genes including stx, eae, and enterohemorrhagicE. coli hly. In addition, all strains were examined for the presence of another genetic element, the high-pathogenicity island (HPI). The HPI was first described in pathogenic Yersiniaspecies and encodes the pesticin receptor FyuA and the siderophore yersiniabactin. The HPI was found in the genome of distinct clonal lineages of STEC, including all 31 eae-positive O26:H11/H⁻ strains and 7 of 12 eae-negative O128:H2/H⁻ strains. In total, the HPI was found in 56 (27.2%) of 206 STEC strains. However, it was absent from the genome of all 37 O157:H7/H⁻, 14 O111:H⁻, 13 O103:H2, and 13 O145:H⁻ STEC isolates, all of which were positive for eae. Polypeptides encoded by the fyuA gene located on the HPI could be detected by using immunoblot analysis in most of the HPI-positive STEC strains, suggesting the presence of a functional yersiniabactin system. The HPI in STEC was located next to the tRNA gene asnT. In contrast to the HPI of other pathogenic enterobacteria, the HPI of O26 STEC strains shows a deletion at its left junction, leading to a truncated integrase geneint. We conclude from this study that theYersinia HPI is disseminated among certain clonal subgroups of STEC strains. The hypothesis that the HPI in STEC contributes to the fitness of the strains in certain ecological niches rather than to their pathogenic potential is discussed.