Paratuberculosis (Johne's disease), caused by Mycobacterium avium subsp. paratuberculosis (MAP) is one of the most common and contagious infections of farm animals mainly cattle herds. MAP leads to a chronic and progressive enteric disease that results poor body condition, reduced milk production, premature culling, and decimate slaughter value in cattle industry. The aim of this study was to investigate of the seropositivity of MAP in cattle with chronic diarrhea in Middle Black Sea Region. For this purpose, a total of 859 Holstein-Friesian dairy cattle (2 years old) from 15 herds with companied to chronic diarrhea were examined in Amasya and Samsun provinces, between 2012 and 2014. MAP antibodies were determined by indirect commercial enzyme-linked immunosorbent assay ELISA (iELISA) kit. Of the 859 samples, 86 (10.0%) and of 15 herds, 7 (46.7%) were positive for the MAP antibodies. In 2012, 2013 and 2014, positive rates of 2.4%, 21.5% and 50% were detected, respectively. As a result, there was a significant increase in MAP seropositivity in chronic diarrheal cattle. It is thought that the results of the study will contribute to the development of effective control programs for eradication of the disease and clinicians working in the region will benefit from the attention of veterinarians.