Mycobacterium avium subspecies paratuberculosis (MAP) infection is considered one of the most wide spread infection in ruminants. MAP infection is important disease that cause great economical losses for dairy industry. Successful control of the disease faces major impediments particularly the long incubation period, low shedding of the organism and low sensitivity of the diagnostic methods in detecting the disease during the subclinical period This study was aimed to estimate the prevalence of John's disease in the dairy herds of eastern province in Saudi Arabia using the newly developed diagnostic tests, ELISA and PCR. A total of 687 serum and fecal samples were collected from dairy cattle of four different ages, one, tow, four and six years age cattle of different dairy farms. Using IDEXX ELISA to screen the serum samples the results indicated 15 (2%) samples were positive and 17 (2.5%) were inconclusive. The results were reconfirmed using another commercial ELISA ID Vet. However due to the budget shortages the PCR test were applied only on 62 ELISA-negative samples that their OD readings were the highest and all the inconclusive samples. The PCR revealed more positive samples (22/62=32%), interestingly among the samples of the tow year old cattle. The study has confirmed conclusively the presence of the disease in the Eastern province dairy herds but the overall prevalence rate could not be drawn due to the limited number of the tested animals. However, the low number of positive samples could indicate low prevalence, but the actual prevalence rate of the infection requires more extensive and elaborate survey using longitudinal approach.