Escherichia coli O157:H7 is a foodborne pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. Positive identification of E. coli O157:H7 is made using biochemical tests and specific antisera or latex agglutination reagents for the O157 and H7 antigens. However, under certain conditions, some E. coli O157:H7 isolates can appear to be nonreactive with H7 antisera and may require multiple passages on motility medium to restore H7 antigenicity. In this study, we compared the performance of a real-time PCR test with that of a method using latex agglutination reagents to detect the presence of the fliC(h7) gene or the H7 antigen, respectively, in E. coli O157:H7 isolates. One hundred twenty-six E. coli strains were tested including reference strains and strains isolated from meat. Lyophilized E. coli O157:H7 isolates were rehydrated and were plated on sheep blood agar without passage on motility medium. All strains were analyzed in parallel by a real-time PCR test targeting the fliC(h7) gene and by a latex agglutination test that detects the H7 antigen. The real-time PCR assay showed 100% agreement with the H7 status reported for reference strains and E. coli O157:H7 meat isolates. The latex agglutination test results agreed with the H7 status reported for the E. coli O157:H7 reference strains and non-O157:H7 strains, except for one, E. coli O117:H7; however, 42% (42 of 100) of the E. coli O157:H7 meat isolates tested negative for the H7 antigen by latex agglutination. The real-time fliC(h7) PCR test can be used to confirm E. coli O157:H7 strains that are not expressing the immunoreactive H7 antigen.