Yersinia enterocolitica is one of the important enteric pathogens which are gram-negative rods that are motile when isolated from environment but become nonmotile in mammalian host which cause human disease due to consumption of contaminated water and food and it has an invasivness ability to cross the gastrointestinal mucosa to infect the underlying tissue The pathogenic Yersinia enterocolitica always harbouring the important virulence factors , Such as the virulence 70Kbp plasmid which encodes the Yop virulon and the HPI which encodes the Yersiniabactin iron responsible genes, but still other virulence genes in Yersinia enterocolitica exists need to be identified and chracterized. Subtractive hybridisation is one of the most powerfull tools for the identification of virulence genes in wide range of bacterial pathogens, and in this study the hybrydization of high pathogenic Yersinia enterocolitica O:8 and low pathogenic Yersinia enterocolitica O:5 was successfull to identify the two novel genes which are probably have relation to virulence. The prepilin peptidase which was proved by PCR in most pathogenic Yersinia species,and it is responsible for the faimbrial and pilli formation which has an adhesive and congugative functions which are important for the genetic materials transfere and the other gene is an invasine Inv homolog sequence which has an ORF of invasine Inv of pathogenic Yersinia enterocolitica which could be named as Inv2