Simple SummaryIn this study, serological and molecular assays in 55 pigs in Sicily were conducted in order to investigate Leptospira spp. prevalence and to carry out strain characterization. A seropositivity rate of 16.4% was determined; 3.64% of kidney samples tested positive for pathogenic Leptospiral DNA. Obtained data showed that Leptospira infection is common among pigs in southern Italy, confirming the importance of Leptospiral infection in pigs and reaffirming the potential role of these animals as a source of infection for humans (occupational risk) and other animals. Our study delivers a comprehensive overview based on up-to-date data to deepen the knowledge of swine leptospiral infections, characterize new potential emerging strains, and reinforce control measures able to reduce the infection risk in swine herds.Leptospirosis is a re-emerging zoonosis of worldwide significance; a wide spectrum of wild and domestic animal species act as natural or accidental hosts. Swine can act as maintenance or accidental hosts of pathogenic Leptospira spp. This study aimed at investigation of Leptospira spp. prevalence and diversity in slaughtered pigs in southern Italy (Sicily). In total, 55 samples of kidneys and blood were collected. Microscopic agglutination test and real-time PCR were performed to detect pathogenic and intermediately pathogenic Leptospira. Partial rpoB gene sequencing and multi-locus sequence typing (MLST) were performed to characterize Leptospira species. The analysis showed a seropositivity rate of 16.4%, with Australis representing the most frequently identified serogroup (63.64%); Pomona and Sejroe were detected with a prevalence of 27.27% and 9.09%, respectively. Pathogenic Leptospiral DNA was detected in 2 kidney samples (3.64%). Leptospira were identified through MLST as L. borgpetersenii serovar Tarassovi (serogroup Tarassovi). Obtained data confirmed the presence of Leptospira infection among pigs in southern Italy, suggesting that management of these animals may be considered an occupational risk for humans.