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Objective : This study purposed to describe the product Polymerase Chain Reaction (PCR) and sequencing of DNA Mycobacterium (M.) tuberculosis from spesimen sputum of tuberculosis (TB) patients in Medan. Material and Methods : Sputum were collected from patients that diagnosed of pulmonary tuberculosis by physician. Specimen were processed by PCR and sequencing. Results : All PCR specimen showed the presence of M. tuberculosis at 126 bp. Sequencing results showed, respectively, the 6 specimen mutation and non mutation of M. tuberculosis. Conclusion : The PCR method is rapid, requires simple equipment, the results are easy to interpret,  low-cost test  and can be used as first line test for rapid detection of M. tuberculosis spesimen in developing countries. Sequencing is a useful tool for early diagnosis and allows choosing the therapy appropriately, and thus reducing the probability of resistance to other drugs during treatment.