Prevalence of Bovine Johne's Disease (BJD) was estimated in dairy cattle herds (Hariana breed) exhibiting weakness and emaciation, using 2 sensitive tests. Cattle belonged to government (DDD Farm) and private dairy farms (Krishna Balram Goshala, Malwiya Goshala, Panchayati Goshala and cows reported at Kothari Veterinary Hospital) in Mathura district (UP) of North India. Serum and fecal samples from 120 cattle were screened by indigenous ELISA kit and fecal culture. ELISA kit, (using protoplasmic antigen from native Mycobacterium avium subsp. paratuberculosis 'Bison type' strain of goat origin), originally developed for goats and standardized in bovines was evaluated for field use in bovine herds (Hariana breed of cattle), exhibiting clinical disease. JD is endemic in animals in this region; therefore, cows in strong positive category were considered sero-positive. IS900 PCR was used to characterize colonies of Mycobacterium avium subsp. paratuberculosis (MAP). Prevalence of MAP was 20.8 and 28.3% by ELISA kit and fecal culture, respectively. There was agreement in 79.1% cases in 2 tests and 14.1 and 6.6% positives were detected exclusively in fecal culture and ELISA, respectively. Sensitive and specificity of ELISA kit was 50.0% and 90.6% respectively and were comparable to commercial kits available for surveys of BJD. Screening of on 224 dairy cattle using this kit as 'herd screening test', sero-prevalence of MAP in dairy herds was 23.6%. Individually, 36.3, 35.0, 16.0, 29.0 and 33.3% cattle were positive from Krishna Balram Goshala, Malwiya Goshala, DDD Farm, Panchayati Goshala and Kothari Veterinary Hospital, respectively. The 229 bp band obtained from amplification of DNA was characteristic and similar to control DNA from MAP 'Bison type' strain of goat origin (characterized previously on the basis of IS 1311 PCR-REA by Sevilla et al., 2005). Of the 34 DNA isolated from colonies in fecal culture, 85.2% were amplified by IS900 PCR. Prevalence of MAP was high in clinically suspected dairy cattle in North India. Indigenous ELISA kit was validated with respect to fecal culture and utility as 'Field Herd-screening test'.