Yersiniosis is the third most reported food-borne zoonosis in Europe. The aim of the present study was to perform the search for Yersinia enterocolitica in food samples collected from Apulia and Basilicata regions (Southern Italy) and to characterize any isolates by classical and modern analytical methods. A total of 130 samples were analyzed between July 2018 and July 2019: most of them were raw milk and dairy products made from it. Furthermore, 8 out of 130 samples were individual milk samples collected from bovines reared in a Brucella-free farm which showed false positive serological reaction for brucellosis due to the presence of pathogenic Y. enterocolitica O:9 biotype 2 in faeces. The Real Time PCR targeting the ail gene and the culture method were performed to detect pathogenic Y. enterocolitica. Isolates were subjected to API 20E (Biomerieux) and MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization Time-of-Flight) for species identification. All samples were negative for the ail gene. The culture method allowed to isolate suspicious colonies from 28 samples. The API 20E system and the MALDI-TOF MS technique identified 20 Y. enterocolitica and 1 Y. intermedia in a concordant way. The remaining 7 strains were all identified as Y. enterocolitica by the API 20E system, while the MALDI-TOF MS recognized 4 Y. intermedia, 1 Y. bercovieri and 2 Y. massiliensis. Genotypic characterization of the discordant strains was performed by rMLST and it confirmed the MALDI-TOF MS' results. Only non-pathogenic Y. enterocolitica biotype 1A strains were found, although with a non-negligible prevalence (P = 0.15 with CI 95% = ± 0.06). This study indicates a poor circulation of pathogenic Y. enterocolitica in food products made and marketed in the investigated areas. However, the small number of samples, insufficient for some food categories such as meat and vegetable, does not allow to exclude the presence of pathogenic strains at all.