The virulence profiles of most non-O157 Shiga toxin (Stx)-producing Escherichia coli (STEC) are unknown. STEC belonging to serogroups O26, O103, and O111 were characterized to determine clonality and to profile virulence traits. Isolates within STEC serogroups O26, O111, and O103 were >80% identical in their randomly amplified polymorphic DNA pattern, suggesting clonality in these serogroups. The serogroups were distantly related to each other and to E. coli O157:H7. stx restriction fragment length polymorphism differentiated some STEC with the same randomly amplified polymorphic DNA pattern. The hemolytic phenotype of STEC O103:H2 isolates resembled that of α- and not enterohemorrhagic E. coli hemolysin. Virulence gene (eae, E-hly, espP, etp, katP, stx) probing demonstrated serogroup-specific profiles. Pathogenic STEC O26, O103, and O111 belong to their own lineages and have unique profiles of virulence traits that are different from the virulence profile of E. coli O157:H7.