Current pathogen typing methods have suboptimal sensitivity and specificity. DNA sequencing offers an opportunity to type pathogens using single nucleotide polymorphisms (SNPs) with greater degrees of discrimination than pulsed-field gel electrophoresis (PFGE) and other methodologies. In a recent cluster of Escherichia coli O157:H7 infections attributed to salad bar exposures and romaine lettuce, a subset of cases denied exposure to either source, though PFGE and multiple-locus variable-number tandem-repeats analysis (MLVA) suggested all isolates had the same recent progenitor. Interrogation of a pre-identified set of 3,442,673 nucleotides in backbone open reading frames (ORFs) identified only 1 or 2 single nucleotide differences in three of 12 isolates from cases who denied exposure. Backbone DNA of nine of nine and three of three cases who reported or were unsure about exposure, respectively, were isogenic. Backbone ORF SNP set sequencing offers pathogen differentiation capabilities that exceed those of PFGE and MLVA.