Non-O157 Shiga toxin-producing Escherichia coli (STEC) are increasingly recognized as foodborne pathogens worldwide. Serogroups O26, O111, and O103 cause most known outbreaks related to non-O157 STEC. Pathogenicity islands (PAIs) play a major role in the evolution of STEC pathogenicity. To determine the distribution of PAIs often associated with highly virulent STECs (OI-122, OI-43/48, OI-57, and high pathogenicity islands) among STEC O26, O103, and O111, a collection of STEC O26 (n=45), O103 (n=29), and O111 (n=52) from humans and animals were included in this study. Pulsed-field gel electrophoresis (PFGE) with XbaI digestion was used to characterize the clonal relationship of the strains. In addition, a polymerase chain reaction-restriction fragment length polymorphism assay was used to determine eae subtypes. Additional virulence genes on PAIs were identified using specific PCR assays, including OI-122: pagC, sen, efa-1, efa-2, and nleB; OI-43/48: terC, ureC, iha, and aidA-1; OI-57: nleG2-3, nleG5-2, and nleG6-2; and HPI: fyuA and irp2. A PFGE dendrogram demonstrated that instead of clustering together with strains from the same O type (O111:H8), the O111:H11 (n=14) strains clustered together with strains of the same H type (O26:H11, n=45). In addition, O26:H11 and O111:H11 strains carried eae subtype β, whereas O111:H8 strains had eae γ2/θ. The O26:H11 and O111:H11 stains contained an incomplete OI-122 lacking pagC and a complete HPI. However, a complete OI-122 but no HPI was found in the O111:H8 strains. Additionally, aidA-1 of OI-43/48 and nleG6-2 of OI-57 were significantly associated with O26:H11 and O111:H11 strains but were almost missing in O111:H8 strains (p<0.001). This study demonstrated that H11 (O111:H11 and O26:H11) strains were closely related and may have come from the same ancestor.