The purpose of this study was to identifying the M. avium paratuberculosis (MAP), investigating its outbreaks and optimizing a fast, accurate and simple method based on PCR and RFLP for detection of this subspecies and their strains. This pathogen cause a chronic infection called Johne's disease. Johne's disease a ruminant chronic infection and economically, is one of the most important disease in livestock industry. 243 fecal samples and 56 raw milk samples of suspected cattle, from some farms of Mashhad were gathered. After DNA extraction, in order to identifying contaminated samples, a specific MAP insertion segment called IS900, with length of 413bp was amplified with PCR using P90 and P91 primers. Then, in order to determining the cow and sheep strains of MAP, 268bp fragment of insertion segment IS1311 was amplified with PCR using M56 and M94 primers. The resulting fragments were digested with HinfI restriction enzyme. 107 of 243 stool samples (44%) and 10 of 56 raw milk samples (18%) were identified as infected with MAP. Among 56 Milk and feces samples taken from the same animal, 19 stools and 10 milk samples were identified as infected (18% vs. 34%). Results of enzymatic digestion also showed that all detected MAP were from bovine isolates.