Objective: The purpose of this study was to isolate and characterize Mycobacterium tuberculosis in Khartoum state using Polymerase Chain Reaction technique. Methodology:71 specimens of sputum were collected from different hospital in Khartoum state were positive with Ziehl Neelsen stain.specimens were decontaminated and  inoculated on Lowenstein Jensen media according to modified Pettrof's  method, biochemical tests, DNA was extracted from each specimen, and then the Polymerase Chain Reaction technique was adopted to detect Insertion Sequence IS6110 gene of M. tuberculosisin these specimens. Results: Out of 71 samples, 57 (80.2%) of the isolates were Mycobacterium tuberculosis complex organisms, biochemical tests showed 68 (95.8%) were sensitive for Para-nitrobenzoic acid; 56 (78.9%) were resistant to Thiophene–2Carboxylic Acid Hydrazide (TCH); 68 (95.8%) were positive for nitrate reduction and 65 (91.6%) were catalase negative at 68°C. while 5(7.1%) revealed no growth. Sixty six Mycobacterium tuberculosis complex isolates were subjected to PCR. Fifty two (78.8%) showed that the positive results while, Fourteen (21.2%) of specimenc were negative by PCR. Conclusion: This results revealed clearly the importance of conventional methods including Z.N stain and culture techniques in the diagnosis of TB especially if there is other invaders like Mycobacteria other than tuberculosis are suspected.