Listeria monocytogenesis 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. monocytogenescan contaminate food and food-processing environments. In particular, ready-to-eat (RTE) products are the most common source associated with listeriosis.L. monocytogenesvirulence 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 ininlAlead to the production of a truncated protein correlated with attenuate virulence. In this study, 849L. monocytogenesisolates, collected from food, food-processing plants, and clinical cases in Italy, were typed and analyzed for the presence of PMSCs in theinlAgene 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 ofinlAPMSC mutations in food and environmental isolates was higher than that in clinical isolates. The results reveal the distribution of the virulence potential ofL. monocytogenescirculating in Italy and could help to improve risk assessment approaches.