Technical Abstract: Cronobacter spp. (formerly Enterobacter sakazakii) and Salmonella are increasingly implicated as important bacterial contaminants in low-moisture food products, including powdered infant formula. Estimates indicate that 40-80% of infants infected with C. sakazakii and/or Salmonella in the U.S. 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 producing virulence factors and genes responsible for unique phenotypes have the potential to function as biomarkers for the detection and identification of Cronobacter spp., Salmonella, and the other foodborne pathogens in low-moisture food products. In this paper, a chitinase gene, a well-studied virulence factor in fungi, plants, and bacteria, was used to confirm this approach to biomarker discovery. We found that the chitinase gene has very low sequence variability and/or polymorphism between Cronobacter spp. and Salmonella while differing significantly in other foodborne pathogens, either by sequence blasting or experimental testing, including PCR amplification and direct sequencing. Among the foodborne pathogens tested, only Salmonella, Citrobacter, and Cronobacter spp. have a similar chitinase or chitinase-like gene, as determined by computational blasting and PCR.