Abstract: Johne’s disease is endemic in the domestic riverine buffalo population of the country and bio-load of Mycobacterium avium subspecies paratuberculosis is increasing in the absence of indigenous diagnostic kits and control programs. A new ‘dot-ELISA kit’ has been developed and validated with indigenous plate ELISA for the screening of buffaloes against Johne’s disease. Out of 156 serum samples screened 41.0 (64), 85.8 (134) and 85.2% (133) were positive for MAP infection by indigenous plate ELISA kit condition (A), condition (B) and indigenous dot ELISA, respectively. Dot-ELISA kit detected 85.2 (133) and 90.3% (141) buffaloes as positive together with indigenous plate ELISA kit in condition A and B, respectively. Comparison of ‘Indigenous plate-ELISA’ with ‘Indigenous dot-ELISA’ revealed substantial agreement between two tests. Study showed that ‘Indigenous dot-ELISA test’ has potential to be sensitive and cost effective ‘Field based herd screening test’ for the large scale screening of the domestic livestock population against Johne’s disease. The study also showed that despite high slaughter rate, incidence of Johne’s disease was high in native population of riverine buffaloes (Bubalus bubalis) and call for immediate control of disease.