Modern molecular methods offer the advantages of simplicity and short time-to-results compared to traditional culture methods. We describe the validation of a new Real-Time PCR method to detect E. coli O157:H7 in five food matrixes. The complete system consists of the MicroSEQ E. coli O157:H7 Detection Kit, sample preparation (two sample preparation methods, the PrepSEQ Nucleic Acid Extraction Kit and the PrepSEQ Rapid Spin Sample Preparation Kit, were validated), the Applied Biosystems 7500 Fast Real-Time PCR instrument, and RapidFinder Express software. The test method was compared to the U.S. Department of Agriculture Microbiology Laboratory Guidebook 5.04 reference method for detecting E. coli O157:H7 in 25 g and 375 g ground beef and beef trim, and to the ISO 16654 reference method for detecting E. coli O157:H7 in 25 g spinach, orange juice, and apple juice. The MicroSEQ E. coli O157:H7 Detection Kit showed equivalent detection compared to the corresponding reference method based on Mantel-Haenszel Chi-square statistics for all matrixes tested. An independent validation confirmed these findings on ground beef. The MicroSEQ kit detected all 51 E. coli O157:H7 strains tested and showed good discrimination against an exclusivity panel of 30 strains.