Enterohemorrhagic Escherichia coli (EHEC), including O157 and non-O157 serotypes are significant foodborne pathogens that require sensitive and discriminatory methods for detection and characterization. There are numerous PCR-based methods for the detection of EHEC virulence factors, but the time and cost involved with large-scale screening efforts and population level analyses have limited the size and scope of studies. Recent technological advancements have combined the high-throughput performance of the microarray with the specificity and sensitivity of real-time qPCR to make large-scale screening efforts both time- and cost-effective. This study identified and evaluated a panel of 28 genetic markers including known virulence and regulatory genes, O-antigen genes, and select prophage regions of O157 and non-O157 EHEC that can be used with high-throughput PCR to virulotype, serotype, and preliminarily subtype large numbers of isolates. The PCR assays for the target genes were shown to be robust using multiple extraction methods and PCR platforms. Preliminary quantitative PCR showed that an EHEC concentration of 10(4) CFU/ml or lower could be detected, with a linear range of detection over five to six orders of magnitude. The panel of 28 target genes has the potential to become an integral tool in outbreak, environmental, and genetic investigations of EHEC.