Objective: Listeria monocytogenes is a food borne pathogen that causes listeriosis. In the environment, the pathogen is most commonly isolated from soil and silage. In foods, it has been found in raw or processed food samples including dairy products. The purpose of this study was to determine the prevalence of raw milk contamination with Listeria genus and Listeria monocytogenes. In this study 100 bulk tank milk samples which were delivered to Pegah pasteurization plant in Mashhad were collected randomly. Method & Materials: For isolation and identification of Listeria spp and Listeria monocytogenes, samples were enriched using cold enrichment method in Listeria enrichment broth, followed by plating out supplemented oxford agar. Colonies with black color and a black haze considered as suspected colonies. Conventional tests including oxidase, catalase, and gram staining and motility test were performed too. For final identification of suspected colonies a multiplex-PCR assay, using primers specific for prs and prf A genes was employed. Results & Conclusion: Using this method, the contamination of raw milk with Listeria spp and Listeria monocytogenes were determined 34% and 4% respectively. To control the accuracy of the m-PCR results, an internal control test, using universal primers specific for 16S rRNA gene, was performed. The specificity of the employed test determined as 100% and its sensitivity was 3.5x103 cfu ml-1. Considering the high specificity and sensitivity of the employed multiplex-PCR assay, it is recommended to use this method for final identification of suspected colonies for Listeria spp and Listeria monocytogenes

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