Escherichia coli are ubiquitous in the world, and for the most part are non-pathogenic and part of the normal lower gastrointestinal tract in mammals. However, some pathogenic isolates can cause severe disease that range from meningitis to hemorrhagic colitis (HC). In recent years, Shiga toxin-containing E. coli (STEC) have been a major cause of food borne and environmental cases of HC and hemolytic uremic syndrome. One STEC serotype, O157:H7, has been responsible for numerous food-associated outbreaks and recalls worldwide. The protocols in this unit will allow the reader to use real-time polymerase chain reaction genotyping to identify isolates that are more likely to cause disease in humans. The genotyping assay targets a single-nucleotide polymorphism (SNP) in the tir gene. The tir gene is located in a virulence operon called the locus for enterocyte effacement and functions as a receptor for the tight adherence of E. coli O157:H7 to epithelial cells. As more genomes are sequenced, informative SNPs that associate with phenotypes will be identified. Identifying isolates not only by their genus and species, but also by using other informative genomic traits will increase the general knowledge about their genetic diversity.