Abstract: The main aim of this study was detection of subclinical forms of Johne’s disease on dairy farms which were suspected of having Johne’s disease or history of it and confirmatory paraclinical of paratuberculosis in them. Also, the isolated bacteria will be used for genotyping and molecular epidemiology of paratuberculosis in the next studies. Samples were gathered of 137 farms in different parts of Razavi Khorasan Province of Iran. There were 505 fresh-fecal and manure samples and 148 raw milk samples including the bulk milk transporters, milk samples of cows with paratuberculosis clinical signs and the farm bulk milk tanks. The samples were cultured on Herrold’s egg yolk medium with and without mycobactin J. The samples, in which the bacteria could merely grow on Herrold’s media containing mycobactin J, were considered to be positive. Additional tests such as colony morphology, acid fast staining and Nested PCR-IS900 assay were performed to confirm isolated bacteria. Diagnostic test of M. paratuberculosis culture was positive in milk, manure and fresh-fecal samples of 29 farms of 137 farms. Out of 653 samples, 50 cases have found positive after cultivation. The number of the bacterial colonies was varied from 1 up to 250. In addition to fresh-fecal samples, using of manure samples and milk samples taken from farm bulk milk tanks can help to detect subclinical forms of Johne’s disease and disease screening in dairy farms. For control of Johne’s disease, mainly subclinical or hidden forms are noticed. In order to achieve this aim, the presences of Mycobacterium avium subsp. Paratuberculosis were investigated in different samples.