Paratuberculosis or Johne's Disease (JD) is a chronic and incurable granulomatous enteric disease affecting cattle, buffaloes, sheep, goats and the other ruminants caused by M. avium subspecies paratuberculosis (MAP). In this study, A total of 300 cows (240 clinically diseased animals and 60 apparently healthy animals) were examined for the presence of clinical signs of johne's disease including incurable chronic diarrhoea, interment firstly then intense and continuous which is not responding to treatment, emaciation and progressive weakness. Fecal and serum samples (each of 300) were collected from the examined cows housed in 3 Egyptian Governorates (Sharkia, Kalyoubia and Giza). Fecal samples were collected then examined according to the pooling procedure and decontaminated by Hexa decylpyridinium chloride solution (HPC 0.9% prior to culturing on Herrold's Egg Yolk Medium (HEYM). MAP was isolated from 34 of the 60 pooled fecal samples tested (57%). Fecal smears were examined using Ziehl- Neelsen stain (ZN) for the presence of acid fast bacilli revealing 29 fecal smears (48%) of 60 fecal smears were positive. ELISA was conducted on serum samples to detect antibodies against MAP, 212 (71%) of serum samples were positive for antibodies against MAP. Molecular confirmation by PCR IS900 assay was carried out using specific primers directly on fecal sample, Out of the 60 pooled fecal samples, 45 pools (75%) were positive. A phylogenetic analysis for determining the genetic difference between current infection and other infected strains in other localities was conducted.