Paratuberculosis is an important disease of ruminants with a worldwide distribution. In developing countries where funding constraints challenge establishment of control schemes, large losses are incurred on cattle farmers due to paratuberculosis. In this study, faecal specimens from Holstein-Friesian cows with progressed and moderate clinical paratuberculosis (N = 223) from 13 dairy farms in Isfahan, Central Iran, were subjected to bacterial culture. Culture growth diagnostic for M. avium subsp. paratuberculosis was found in cattle from nine of the 13 farms and in 71 of the cattle studied. These results illustrate the emergence of PTb in this region, and they imply that PTb should be given a higher priority for veterinary measures.