Background: Tuberculosis is a critical infectious disease primarily affecting the lungs and is more common in developing countries. In the 21st century, it forms a significant problem for world public health especially with the emergence and rising of drug resistant TB. Microbiological methods are the clue for the laboratory diagnosis. The ordinary methods for TB identification showed either weak sensitivity as in microscopy or lateness for many weeks as in culture. The evolution in molecular biology gives a chance for fast diagnosis of Mycobacterium tuberculosis helping start proper treatment early and holding its spread. The initial critical step in PCR is DNA extraction. Objective: The aim to evaluate different extraction methods of Mycobacterium tuberculosis retrieved directly from sputum samples and from LJ isolates from same patients and comparing DNA yield using conventional PCR. Methodology: DNA from 32 sputum samples from TB patients extracted by solid, digestion and phenol extraction methods, DNA from 40 LJ isolates extracted by solid, boiling and Cetyl trimethylammonium bromide methods. Extracted DNA was evaluated by conventional PCR. Results: Among 32 sputum samples, the extracted DNA by phenol method was 21/32 (65.62%) with highest DNA yield, digestion method 14/32 (43.75%) and solid phase method 1/32 (2.5%) with least DNA yield. From 40 MTB LJ culture isolates, the extracted DNA by boiling method was 28/40 (70%), CTAB method 18/40 (45%) and solid phase method 2/40 (5%). Conclusion: Phenol method was the best method (mean rank 2.34) for DNA extraction from sputum samples, while the easy and economic boiling method was the best method (mean rank 2.45) for DNA extraction from LJ culture isolates. The worst method of DNA extraction from both sputum and culture was phase solid method. A greater and easier yield of DNA was obtained from MTB LJ Culture than sputum.