Listeria monocytogenes and L. ivanovii are important food-pathogens for human and animal. The diagnostic of Listeria in food using culture medium requires time and laborwork, because there are many other nonpathogenic species like L. innocua, L. welshimeri, L. seeligeri and L. grayi in Genus Listeria. For these reasons, Lismix multiplex PCR method was developed as a rapid method for the detection and identification of Listeria. In this study we developed a practical system of Lis-mix PCR detection for the application to food samples and new developed Siw-mix III PCR system. Using this Lis-mix PCR system overall 69 listerial strains were successful species-identified and confirmed. Also, the Siw-mix III PCR system allows the species-specific identification among L. ivanovii, L. welshimeri and L seeligeri in a single PCR.