Prevalence of Mycobacterium avium subsp. paratuberculosis (MAP) has been studied in cattle (milk samples) and buffaloes (tissue samples). Samples were screened using culture and IS900 PCR (tissues, milk fat and milk sediment), and ELISA (milk and serum). Prevalence of MAP was 78.0% by milk culture (fat - 60.0% and sediment - 50.0%). Milk ELISA was standardized using MAP (Bison type) of goat origin. Milk ELISA detected 32.0% cows as positive from 115 milk samples from dairies. These samples were also screened using MAP (bovine type) antigen procured from USA. Serum samples were also screened using serum ELISA and both the antigens. Molecular characterization revealed that most of the isolates were Bison type and only one isolate was bovine type. High prevalence of MAP in the milk of lactating cows pose direct threat to humans and young calves alike. Raw milk is still consumed as medicine in some parts of rural India. Tissues (mesenteric lymph nodes and large intestine) from 50 slaughtered buffaloes were screened by culture and IS900 PCR. Of these 48.0% buffaloes were culture positive (MLN - 34.0% and LI - 36.0%). Serum ELISA test was standardized using MAP bison type (native) of goat origin and bovine type (USA), antigens. Of the 167 serum samples 46.7% buffaloes positive in plate ELISA. While none of the buffalo w detected as positive in plate ELISA using MAP bovine type antigen. MAP isolated were typed as MAP bison type on the basis of IS 900 and IS 1311 PCR - REA analysis.