Molecular methods involving nucleic acids have become an indispensable part of any scientific study in the modern era. Towards this, the isolation of DNA/RNA from the organisms is a crucial step where the purity and quantity need to be high to arrive at a meaningful result. In this study, three DNA extraction procedures have been tried with the primary aim to find its suitability in genotypic analysis of Mycobacterium tuberculosis. A total of 104 isolates of M.tuberculosis from solid culture were subjected to Boiling method, CTAB method, and a commercial kit based system and the extracted DNA was analyzed for the amount and the purity. The highest DNA concentration resulted from the commercial extraction method (value 89.09 µg/ml ± 14.69), while the lowest one resulted from the CTAB method (value 35.62 µg/ml ± 8.4); while the yield from boiling method was higher compared to the CTAB method (51.67 µg/ml ± 9.21). Spectrophotometric analysis for A260/280 ratio showed that purity of DNA was high in CTAB method compared to remaining two extraction methods. The extracted DNA by the three methods all showed distinct bands when amplified with MIRU20 primer. Culture of material after heat extraction was cultured on solid medium to check the viability, and out of 104 samples, a total of 4.3% remained viable. Thus the boiling method is a fast and reliable means of extracting DNA for genetic analysis of M.tuberculosis, but a small number of isolates which escape the killing remains a matter of concern.