Mycobacterium avium subsp. paratuberculosis is a pathogen that causes johne’s disease in animals and is implicated in Crohn’s disease in humans. Culture of Mycobacterium avium subsp. paratuberculosis (MAP) from faeces has been considered the gold standard for the diagnosis of paratuberculosis for many years. However, direct faecal polymerase chain reaction (PCR) is becoming more widely used, demonstrating similar sensitivity and specificity to culture. In the present study, faecal culture and IS900 Polymerase chain reaction (PCR) assay of faecal samples was done on 200 clinically suspected cases of Johne’s disease in dairy cattle. One isolates appeared only on the mycobactin J supplemented media at 8–16 weeks post-inoculation. A total of 7 faecal samples out of 200 samples were detected positive by IS900 PCR assay for Mycobacterium avium subsp. paratuberculosis (MAP) yielding an expected product of size 229 bp. The sensitivity of the IS900 PCR was assessed by making ten fold serial dilutions of the known concentration (5 ng/µl) of the standard genomic DNA of MAP. The detection limit of the IS900 PCR was upto5 pg/µl.