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<i>Listeria monocytogenes</i> 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. <i>L. monocytogenes</i> can contaminate food and food-processing environments. In particular, ready-to-eat (RTE) products are the most common source associated with listeriosis. <i>L. monocytogenes</i> 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 <i>inlA</i> lead to the production of a truncated protein correlated with attenuate virulence. In this study, 849 <i>L. monocytogenes</i> isolates, collected from food, food-processing plants, and clinical cases in Italy, were typed and analyzed for the presence of PMSCs in the <i>inlA</i> 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 <i>inlA</i> PMSC mutations in food and environmental isolates was higher than that in clinical isolates. The results reveal the distribution of the virulence potential of <i>L. monocytogenes</i> circulating in Italy and could help to improve risk assessment approaches.