The presence of virulence genes, encoding enterohemorrhagic Escherichia coli (EHEC)-hemolysin (EHEC-hlyA), intimin (eae), and Shiga toxins 1 (stx1) and 2 (stx2), in 178 isolates of pathogenic E. coli, was determined using the polymerase chain reaction with primers specific for each virulence gene. The tested organisms were 120 isolates of E. coli O157:H7 from human patients, cattle, sheep and foods, 16 non-O157:H7 EHEC isolates from patients suffering from hemorrhagic colitis or hemolytic uremic syndrome, 15 non-O157:H7 Shiga toxin-producing E. coli (STEC) isolates from cattle and foods, 26 isolates of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC), and an E. coli K12 strain. Results revealed that all isolates of O157:H7 carried EHEC-hlyA, eae, and one or both stx genes; 15 of the 16 non-O157:H7 EHEC isolates had EHEC-hlyA, but all possessed eae and one or both stx genes; only seven of the 15 non-O157 STEC isolated from cattle and foods contained both EHEC-hlyA and eae genes. The EPEC, EIEC, ETEC, and the E. coli K12 strain did not carry these virulence genes, except eight EPEC isolates were positive for eae. Results suggest that a combination of EHEC-hlyA and eae genes could serve as markers to differentiate EHEC from less pathogenic STEC, and other pathogenic or non-pathogenic E. coli.