Significant phenotypic diversity was observed when we examined the abilities of a number of Cronobacter sakazakii natural isolates to cope with various sublethal stress conditions (acid, alkaline, osmotic, oxidative, or heat stress). Levels of catalase activity and use of acetate as a carbon source, phenotypes commonly used as indirect assays to predict RpoS function, revealed a high correlation between predicted RpoS activity and tolerance to acid, alkaline, osmotic, and oxidative treatments. The rpoS genes were sequenced and analyzed for polymorphisms. Loss-of-function mutations were found in two strains; C. sakazakii DPC 6523 and the genome-sequenced strain C. sakazakii ATCC BAA-894. The complementation of these strains with a functional rpoS gene resulted in an increase in bacterial tolerance to acid, osmotic, and oxidative stresses. The pigmentation status of strains was also assessed, and a high variability in carotenoid content was observed, with a functional rpoS gene being essential for the production of the characteristic yellow pigment. In conclusion, the evidence presented in this study demonstrates that rpoS is a highly polymorphic gene in C. sakazakii, and it supports the importance of RpoS for the tolerance under stress conditions that C. sakazakii may encounter in the food chain and in the host during infection.