Salmonella enterica subsp. enterica is the leading cause of bacterial food-borne disease in the United States. Molecular subtyping methods are powerful tools for tracking the farm-to-fork spread of food-borne pathogens during outbreaks. In order to develop a novel multilocus sequence typing (MLST) scheme for subtyping the major serovars of S. enterica subsp. enterica, the virulence genes sseL and fimH and clustered regularly interspaced short palindromic repeat (CRISPR) loci were sequenced from 171 clinical isolates from nine Salmonella serovars, Salmonella serovars Typhimurium, Enteritidis, Newport, Heidelberg, Javiana, I 4,[5],12:i:-, Montevideo, Muenchen, and Saintpaul. The MLST scheme using only virulence genes was congruent with serotyping and identified epidemic clones but could not differentiate outbreaks. The addition of CRISPR sequences dramatically improved discriminatory power by differentiating individual outbreak strains/clones. Of particular note, the present MLST scheme provided better discrimination of Salmonella serovar Enteritidis strains than pulsed-field gel electrophoresis (PFGE). This method showed high epidemiologic concordance for all serovars screened except for Salmonella serovar Muenchen. In conclusion, the novel MLST scheme described in the present study accurately differentiated outbreak strains/clones of the major serovars of Salmonella, and therefore, it shows promise for subtyping this important food-borne pathogen during investigations of outbreaks.