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Listeria monocytogenes is a widespread Gram-positive pathogenic bacterium that causes listeriosis, a rather rare but severe foodborne disease. Pregnant women, infants, the elderly, and immunocompromised individuals are considered particularly at risk. L. monocytogenes can contaminate food and food-processing environments. In particular, ready-to-eat (RTE) products are the most common source associated with listeriosis. L. monocytogenes virulence factors include internalin A (InlA), a surface protein known to facilitate bacterial uptake by human intestinal epithelial cells that express the E-cadherin receptor. Previous studies have demonstrated that the presence of premature stop codon (PMSC) mutations naturally occurring in inlA lead to the production of a truncated protein correlated with attenuate virulence. In this study, 849 L. monocytogenes isolates, collected from food, food-processing plants, and clinical cases in Italy, were typed and analyzed for the presence of PMSCs in the inlA gene using Sanger sequencing or whole-genome sequencing (WGS). PMSC mutations were found in 27% of the isolates, predominantly in those belonging to hypovirulent clones (ST9 and ST121). The presence of inlA PMSC mutations in food and environmental isolates was higher than that in clinical isolates. The results reveal the distribution of the virulence potential of L. monocytogenes circulating in Italy and could help to improve risk assessment approaches.