The “O157:H7 complex” is comprised mostly of enterohemorrhagic Escherichia coli (EHEC) strains, with serotype O157:H7 being the prototypic and predominate pathogenic strain in the complex. However, several phenotypic O157:H7 variants and genetically closely related O55:H7 serotype enteropathogenic E. coli (EPEC) are also included in the complex. The EHEC strains in the complex share many of the same virulence factors, but can exhibit diverse phenotypic profiles. As a result, identification of the various EHEC strains in the “complex” often requires multiple assays. Using PCR primers that are specific for four characteristic EHEC virulence genes (stx ₁, stx ₂, γ-eae, and ehxA) and to a single nucleotide polymorphism (+92 uidA) gene marker that is highly conserved among strains in the complex, a multiplex PCR assay was developed that simultaneously detects these five markers and allows the identification of the pathogenic EHEC strains in the O157:H7 complex.