Infections with Shiga toxin-producing Escherichia coli (STEC) result in frequent cases of sporadic and outbreak-associated enteric bacterial disease in humans. Classification of STEC is by stx genotype (encoding the Shiga toxins), O and H antigen serotype, and seropathotype (subgroupings based upon the clinical relevance and virulence-related genotypes of individual serotypes). The espZ gene is encoded in the locus of enterocyte effacement (LEE) pathogenicity island responsible for the attaching and effacing (A/E) lesions caused by various E. coli pathogens (but not limited to STEC), and this individual gene ( approximately 300 bp) has previously been identified as hypervariable among these A/E pathogens. Sequence analysis of the espZ locus encoded by additional STEC serotypes and strains (including O26:H11, O121:H19, O111:NM, O145:NM, O165:H25, O121:NM, O157:NM, O157:H7, and O5:NM) indicated that distinct sequence variants exist which correlate to subgroups among these serotypes. Allelic discrimination at the espZ locus was achieved using Light Upon eXtension real-time PCR and by liquid microsphere suspension arrays. The allele subtype of espZ did not correlate with STEC seropathotype classification; however, a correlation with the allele type of the LEE-encoded intimin (eae) gene was supported, and these sequence variations were conserved among individual serotypes. The study focused on the characterization of three clinically significant seropathotypes of LEE-positive STEC, and we have used the observed genetic variation at a pathogen-specific locus for detection and subtyping of STEC.