Cronobacter sakazakii (Enterobacter sakazakii) is an emerging pathogen that has been isolated from powdered infant formula and associated with outbreaks of infection in infants in neonatal intensive care units. In a previous study, we observed that neonatal CD-1 mice are susceptible to C. sakazakii infection and that the pathogen invades brain, liver, and cecum tissues. The study objective was to compare the virulence of three strains of C. sakazakii in neonatal CD-1 mice. The strains tested were MNW2 (a food isolate), SK81 (a clinical isolate), and 3290 (a clinical isolate). Timed-pregnant CD-1 mice were allowed to give birth on gestation day 19 or 20. Neonatal mice were sexed and culled to 10 per litter, each having five males and five females. Neonates were orally gavaged with C. sakazakii strains MNW2, SK81, or 3290 at doses ranging from 10(2.8) to 10(10.5) CFU on postnatal day 3.5. Pups surviving to postnatal day 10.5 were euthanized, and brain, liver, and cecum tissues were excised. C. sakazakii was isolated from all three tissues in mice treated with C. sakazakii, regardless of strain. C. sakazakii strain 3290 was significantly more invasive in brains (42.1% of mice) than were strains MNW2 (6.7%) and SK81 (15.9%). Mortality was observed for all strains of C. sakazakii tested, with SK81 being significantly more lethal (5.6%) than MNW2 (1.2%) or 3290 (0.6%). Our findings suggest that invasiveness does not necessarily correlate with mortality among different strains of C. sakazakii, and the clinical isolates are more virulent than the food isolate.