We identified the cytolethal distending toxin V (CDT-V) gene cluster in 19 (4.9%) of 391 enterohemorrhagic Escherichia coli O157:H7. cdt-V+ strains belonged to five phage types (PTs) and were most frequent within PTs 14 and 34. CDT-V was expressed in all but two cdt-V+ strains and was lethal to cultured endothelial cells. Subtyping schemes should include cdt-V as a marker to differentiate E. coli O157:H7 even within the same phage type.