ABSTRACT Listeria monocytogenes utilizes internalin A (InlA; encoded by inlA ) to cross the intestinal barrier to establish a systemic infection. Multiple naturally occurring mutations leading to a premature stop codon (PMSC) in inlA have been reported worldwide, and these mutations are causally associated with attenuated virulence. Five inlA PMSC mutations recently discovered among isolates from France and the United States were included as additional markers in our previously described inlA single-nucleotide polymorphism (SNP) genotyping assay. This assay was used to screen >1,000 L. monocytogenes isolates from ready-to-eat (RTE) foods ( n = 502) and human listeriosis cases ( n = 507) for 18 inlA PMSC mutations. A significantly ( P < 0.0001) greater proportion of RTE food isolates (45.0%) carried a PMSC mutation in inlA compared to human clinical isolates (5.1%). The proportion of L. monocytogenes with or without PMSC mutations in inlA was similar among isolates from different RTE food categories except for deli meats, which included a marginally higher proportion ( P = 0.12) of isolates carrying a PMSC in inlA . We also analyzed the distribution of epidemic clone (EC) strains, which have been linked to the majority of listeriosis outbreaks worldwide and are overrepresented among sporadic cases in the United States. We observed a significant ( P < 0.05) overrepresentation of EC strains in deli and seafood salads and a significant ( P < 0.05) underrepresentation of EC strains in smoked seafood. These results provide important data to predict the human health risk of exposure to L. monocytogenes strains that differ in pathogenic potential through consumption of contaminated RTE foods.