Mycobacterium avium Subsp. paratuberculosis (MAP) is a gram-positive, small, acid-fast bacillus with high environmental resistance. In animals, especially ruminants, it leads to Paratuberculosis (PTB) or Johne's disease, which is chronic granulomatous enteritis. This bacterium as the main causative agent of Crohn's disease can be a serious threat to human health. This study aimed to detect MAP in pasteurized milk samples produced in Khorasan Razavi province, Iran, using Direct Nested PCR, PCR, and culture methods. In this study, 544 milk samples from Pasteurized Milk Production Companies were selected randomly during the 3-month period. DNA was extracted from milk fat after centrifugation. In order to identify the bacteria, Direct Nested PCR and PCR tests were applied using IS900 and f57, respectively. Furthermore, to detect viable MAP, positive samples resulted from Direct Nested PCR assays were cultured on Herrold's egg medium. For identification of mycobacterial isolates all colonies were processed by PCR based on f57. A total of 544 pasteurized milk samples were assayed, and Mycobacterium paratuberculosis was detected in 39% of them by IS900 Nested PCR, and only 4. 9% of samples were positive in culture method. All the colonies were positive for the f57using PCR. The results of this study indirectly indicated a high level of contamination of pasteurized milk to Mycobacterium paratuberculosis which is due to the large number of affected animals in livestock farms in Khorasan Razavi province. However, in comparison with the other researches, the low percentage of viable bacteria in pasteurized milk can be due to changes in temperature and time in pasteurizing systems of milk production companies in Khorasan Razavi province, Northeast of Iran.