Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne’s disease is a degenerative chronic granulomatous disease of bovines. In the present study, quantitative real time polymerase chain reaction (qRT-PCR) using TaqMan chemistry targeting the IS900 sequence of Mycobacterium avium subsp. paratuberculosis (MAP) was employed for the molecular diagnosis of the disease in bovine faecal samples. Out of 200 bovine faecal samples processed, 7 samples were tested as positive by IS900 qRT-PCR. The sensitivity limit of detection of MAP DNA in faecal samples by qRT-PCR TaqMan assay was found to be 0.05pg. No amplification was observed in other Mycobacterial spp. viz. M. phlei, M. smegmatis, M. intracellulare and M. kansasii.