Low sensitivity of the PCR reaction for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in tissue, fecal, milk and blood samples is attributed to the false negative results primarily due to unsuccessful isolation of DNA and presence of PCR amplification inhibitors in the samples. Therefore, selection of a suitable protocol for the isolation of DNA is crucial for high sensitivity in PCR based detection tests. Study aimed to compare 4 methods of DNA isolation and determined most suitable procedure for isolation of good quality MAP DNA from intestinal tissue samples that would be suitable for IS900 based PCR detection of MAP infection. A total of 25 intestinal tissues (near ileocaecal junction) were collected from farm goatherds endemic for Johne's disease (multi-bacillary colonies on fecal culture and with typical intestinal corrugation). Tissues were homogenized, suspended in sterilized distilled water and supernatant was processed for culture on HEY medium as well as DNA isolation by four different methods. Method 1: DNA was isolated from tissues by method of van Soolingen et al (1991). Method 2: Supernatant of grounded tissues was treated with 0.9% HPC (Hexadecylpyridium chloride) over night and then DNA was extracted using method of van Soolingen et al (1991). Method 3: Supernatant of grounded tissues was treated with tissue lysis buffer (2% Triton-X, 1% SDS, 100mM NaCl, 10mM Tris HCl) [pH 8.0] and proteinase K (20 mg/ml), for proper digestion and afterwards DNA was extracted using phenol and chloroform-isoamyl (24:1) process. Method 4: Samples were first treated with 0.9% HPC as in method 2 and then DNA was isolated as per the method described in Method 3. Isolated genomic DNA was further purified by commercial kit and subjected to IS 900 PCR. For the further confirmation of amplicons, these were subjected to sequencing and BLAST. Using the first method of DNA isolation, 15 (60%) samples were positive. Using method 2, 18 (72.0%) samples were positive, as compared to 13 (52%) samples positive in each of the method 3 and 4, respectively. Amplification of DNA extracted from 25 intestinal tissues by four different methods indicated that Method 2, (treatment of the samples first with 0.9% HPC overnight and subsequent DNA isolation by method of van Soolingen et al., 1992), was more efficient in extracting good quality DNA as compared to other 3 methods. Therefore method 2, as standard method for isolation of DNA from MAP culture may also be adopted for necropsy tissues for high sensitive detection of MAP using PCR based tests. However, results of culture on Herrold's egg yolk medium using same fraction of tissues samples showed 100.0% sensitivity in detecting MAP bacilli.