Diagnosis of tuberculosis is still an ongoing problem however nucleic acid amplification test has emerged as a promising tool for its diagnosis. Single gene target may result in false negative due to the absence or the presence of only few copies of target DNA in some M.tuberculosis isolates. The objective of this study was to evaluate a multiplex PCR (MPCR) for the detection of M. tuberculosis in sputum samples.150 clinically suspected cases of pulmonary tuberculosis were processed for detection of mycobacterial infections by ZN staining smear examination, LJ culture and Multiplex PCR tests which comprised of genus specific primer targeting insertion sequence 6110 (IS6110), MPB64 (23kDa) and Protein b (38kDa). Multiplex PCR showed highest sensitivity of 100 %, followed by 65.2 % for AFB smear when LJ culture was considered as gold standard. Conclusion: Multiplex PCR increased the sensitivity and it can be used to detect samples with M.tuberculosis strains lacking IS6110.