Abstract: Mycobacterium paratuberculosis (MAP) is the cause of John’s disease or paratuberculosis. This is economically one of the important infection diseases in cattle and ruminant industries. This disease is manifested as granulomatosis entrocolitis, lymphadenitis and inflammation local lymphatic vessels. The typical sign of this disease is the progressive loss of patient weight. Considering the importance of detection of this disease in this study, two methods, culture and PCR were used for the identification of this microorganism. In this study 100 milk samples from apparently healthy cows and 100 milk samples from cows that have been suspicious of John's disease were taken from cattle Sarab region. Direct microscope observation after ziehl-neelsen staining was done. Then, bacterial culture on specific medium was carried out and finally, identification of Mycobacterium avium paratuberculosis was examined using PCR and specific primers. Using direct observation, culture and PCR analyses showed that from 100 healthy cow milk samples, 8, 9 and 12 samples were positive MAP for each method, respectively. The results of direct observation, culture and PCR analyses on affected cows were 15, 40 and 44, respectively. The results of this study showed that culture and PCR analyses methods are important in the identification of the causes of this disease. Therefore, considering the frequency of the disease in the studied region, either of those methods can be used in the microorganism identification.