Escherichia coli O157:H7 is an important foodborne pathogen, and foods of bovine origin and fresh produce have been linked to outbreaks. Real-time multiplex PCR assays were developed to detect E. coli O157:H7 in different foods. Apple cider and raw milk (25 ml) and ground beef and lettuce (25 g) were inoculated with 2 or 20 colony-forming units (CFU) of E. coli O157:H7 380-94 and subjected to enrichment in RapidChek E. coli O157:H7 broth at 42°C. One milliliter of the enrichments was removed at 8 and 20 h, and following DNA extraction, real-time multiplex PCR assays targeting the stx1, stx2, and wzyO157 genes in combination with probes and primers targeting either the fliCh7 or the eae genes were performed using OmniMix HS beads and the SmartCycler. The sensitivity of the real-time multiplex PCR assay was about 225 CFU/PCR. E. coli O157:H7 was detected (fluorescent signal generated for all gene targets) in apple cider, raw milk, lettuce and ground beef samples inoculated with 2 or 20 CFU/g or 25 ml after both 8 and 20 h of enrichment. Enrichments of uninoculated food samples were negative using the multiplex PCR targeting the stx1, stx2, wzyO157, and eae genes; however, using the assay targeting the stx1, stx2, wzyO157, and fliCh7 gene combination, a positive result was always obtained for the fliCh7 gene using uninoculated ground beef enrichments. Use of other primer sets targeting the fliCh7 gene gave similar results. The real-time multiplex PCR assays targeting the stx1, stx2, eae, and wzyO157 or the fliCh7 genes are sensitive and specific and can be used for the detection of E. coli O157:H7 in food, except that the fliCh7 gene may not be a suitable target for the detection of E. coli O157:H7 in ground beef.