In Europe yersiniosis related to Yersinia enterocolitica is the third most numerously reported zoonoses. In Italy, notification of yersiniosis is not compulsory; thus, no true incidence rates are available from this country. Yersinia species are ubiquitous and they are also isolated from a wide variety of foods. The objectives of this work were to study a multiplex PCR that could be applicable for a screening of food samples for pathogenic Y. enterocolitica, to characterize strains isolated from human patients, swine carcasses, meat, vegetables, burrata cheese and wild boars collected in Umbria and Marche Regions during 2004-2011, to investigate the detection of genotypic virulence markers ail, ystA, ystB, myfA and hreP and to characterize the recovered isolates by PFGE and MLVA. Among 120 swine carcasses investigated Y. enterocolitica were isolated from 25,8% of the samples and 2 of them were identified as Y. enterocolitica biotype 4 showing all the virulence factors tested and identical PFGE and MLVA profiles. Ten pathogenic Y. enterocolitica strains, recovered from stools of patients, exhibited the genotype ystA+ yadA+ myfA+ hreP+, 6 showed the genotype ystA+ yadA- myfA+ hreP+ and 2 were biotype 1A ystB+. Most of the colonies isolated from the other sources was nonpathogenic and harboured the ystB gene. The multiplex PCR method was effective, fast and simple, capable of detecting pathogenic Yersinia enterocolitica in 48-72 hours. It offers significant advantages over the microbiological methods of isolation, allowing shorter response times and early detection of strains carrying the pathogenicity factor.