ABSTRACTCronobacterspp. (formerlyEnterobacter sakazakii) andSalmonellaspp. are increasingly implicated internationally as important microbiological contaminants in low-moisture food products, including powdered infant formula. Estimates indicate that 40 to 80% of infants infected withCronobacter sakazakiiand/orSalmonellain the United States may not survive the illness. A systematic approach, combining literature-based data mining, comparative genome analysis, and the direct sequencing of PCR products of specific biomarker genes, was used to construct an initial collection of genes to be targeted. These targeted genes, particularly genes encoding virulence factors and genes responsible for unique phenotypes, have the potential to function as biomarker genes for the identification and differentiation ofCronobacterspp. andSalmonellafrom other food-borne pathogens in low-moisture food products. In this paper, a total of 58 uniqueSalmonellagene clusters and 126 unique potentialCronobacterbiomarkers and putative virulence factors were identified. A chitinase gene, a well-studied virulence factor in fungi, plants, and bacteria, was used to confirm this approach. We found that the chitinase gene has very low sequence variability and/or polymorphism amongCronobacter,Citrobacter, andSalmonella, while differing significantly in other food-borne pathogens, either by sequence blasting or experimental testing, including PCR amplification and direct sequencing. This computational analysis forCronobacterandSalmonellabiomarker identification and the preliminary laboratory studies are only a starting point; thus, PCR and array-based biomarker verification studies of these and other food-borne pathogens are currently being conducted.