Raw milk samples of 503 individual buffaloes were collected from dairy farms located in Agra and Mathura cities in North India. Multiple tests (Indirect Fluorescent Agglutination test (i_FAT), IS900 PCR, Microscopy, Indigenous ELISA kit (i_ELISA), Dot-ELISA (d_ELISA) and Latex agglutination test (LAT)) based bio-load and bio-type profile of Mycobacterium avium subspecies paratuberculosis (MAP) was studied. Cumulatively average bio-load was 61.2 percent using three antigen and three antibody based. In i_FAT, IS900 PCR and microscopy, 43.5, 13.3 and 40.9 percent milk were positive for MAP, respectively. Whereas, 32.8, 49.3 and 44.1 percent milk samples were positive in i_ELISA, d_ELISA and LAT, respectively. Bio-typing of representative milk samples using IS900 PCR positive raw milk (67), 13.4 percent were infected with 'Indian Bison Type' biotype using IS1311 PCR_REA. Study concluded that 'Indian Bison type' was the predominant bio-type infecting lactating buffaloes of this region. Raw milk was highly convenient sample in buffaloes and 'milk samples' were first time screened without initial processing of milk samples. Detection limits of each tests was improved. Results of five tests (d_ELISA, LAT, i_ELISA, microscopy, i_FAT were comparable, except IS900 PCR. High bio-load of MAP in milk of buffaloes was major health hazard for human health. High bio-load of MAP was alarming and calls for initiation of Johne's disease control programs in the country.