Escherichia coli O157:H7, a Shiga toxin-producing E. coli, has been the causative agent of many cases of severe, often life-threatening foodborne illness. Because of the importance of E. coli O157:H7 to public health, many molecular typing methods have been developed to determine its transmission routes and source of infection during epidemiological investigations. Pulsed-field gel electrophoresis (PFGE) is currently used by public health organizations to track infections of E. coli O157:H7 and other foodborne pathogens. In this study, we compared the ability of PFGE, multilocus sequence typing (MLST), and repetitive-element PCR (Rep-PCR) to distinguish among 92 E. coli O157:H7 isolates from cattle, food, and infected humans. Several virulence genes, including the intimin gene (eaeA), the hemolysin gene (hlyA), and the H7 fimbrial gene (fliC), and a housekeeping gene for beta-glucuronidase (uidA) were included in MLST. Rep-PCR reactions were performed using a commercially available typing kit (Bacterial Barcodes Inc., Houston, Tex.) with the provided Uprime-RI primer set. Results of the study indicated that PFGE provided the most discrimination among the techniques, identifying 72 distinct PFGE profiles for the isolates; Rep-PCR elucidated 14 different profiles, whereas MLST generated five profiles. Additionally, there did not appear to be any correlation among the typing methods examined in this study. Therefore, to date, PFGE remains the technique of choice for molecular subtyping of E. coli O157:H7.