Listeriosis results from exposure to the foodborne pathogen Listeria monocytogenes. Although many different strains of L. monocytogenes are isolated from food, no definitive tests currently predict which isolates are most virulent. The objectives of this study were to address two major data gaps for risk assessors, variability among L. monocytogenes strains in pathogenicity and virulence. Strains used in our monkey clinical trial or additional food isolates were evaluated for their virulence and infectivity in mice. All strains were equally pathogenic to immunocompromised mice, causing deaths to 50% of the population 3 days after exposure to doses ranging from 2 to 3 log CFU. Doses resulting in 50% deaths on the fifth day after administration were 1 to 2 log lower than those on the third day, indicating that the full course of pathogenicity exceeds the 3-day endpoint in immunocompromised mice. Three strains were chosen for further testing for their virulence and infectivity in liver and spleen in normal (immunocompetent) mice. Virulence was not significantly different (P > 0.05) among the three strains, all resulting in deaths to 50% of mice at 5 to 7 log CFU by 5 days after administration. All strains were equally infective in liver or spleen, with higher numbers of L. monocytogenes directly correlated with higher doses of administration. In addition, there was no preference of organs by any strains. The lack of strain differences may reflect the limitation of the mouse model and suggests the importance of using various models to evaluate the pathogenicity and virulence of L. monocytogenes strains.