Aims: In this study, 105 Listeria monocytogenes strains isolated from humans, foods and environmental samples were characterized using several typing methods. Moreover, serotyping procedure was evaluated, and a cost-effective methodological approach based on preliminary PCRs screening was proposed. Methods and Results: The isolates were analysed by conventional serotyping, multiplex-PCRs for serogroup and lineage identification and PCR-RFLP of inlA gene to identify potentially noninvasive L. monocytogenes. Among the strains, only the serotypes 1/2a, 1/2c, 1/2b, 4b and 3a were identified. The isolates were classified into serogroups I (58 center dot 10%), II (22 center dot 85%), III (12 center dot 38%) and IV (6 center dot 67%). Among clinical strains, lineage I was more represented (68 center dot 75%) than lineage II; whereas, lineage II was more associated with food (90 center dot 24%) and environmental (85 center dot 72%) isolates. Most of food (89 center dot 02%) and environmental (85 center dot 71%) isolates were classified into truncated InlA profiles, whereas the 93 center dot 75% of clinical strains were associated with a complete form of the protein. Conclusion: Molecular techniques were sensitive and specific for classifying strains into serogroup and lineage and in agreement with the serotyping. Moreover, a preliminary PCRs-based screening was proposed to select only the necessary antisera by a flow chart; this methodological approach allows cost saving up to 42%. Our results further suggest the role of InlA protein in human listeriosis, particularly in immunocompetent individuals, and a correlation between truncated protein and serotype. Significance and Impact of the Study: This study further validates molecular methods for L. monocytogenes analysis and proposed a new cost-effective approach for serotyping. It could help to improve a national surveillance network for L. monocytogenes infections in Italy.