Mycobacterium bovis (M. bovis) is the major cause of gastrointestinal tuberculosis in humans. The study was conducted in Lahore to compare 100 blood and 100 sputum samples from suspected patients of active tuberculosis, using the Duplex PCR and conventional methods for the detection of M. bovis and Mycobacterium tuberculosis (M. tuberculosis). In M. tuberculosis, pncA gene and species-specific 500-bp fragment of M. bovis was targeted in Duplex PCR. In sputumsamples 37 % revealed the presence of M. tuberculosis while 05 % samples were positive for M. bovis using duplex PCR. Blood samples, 39 % and 04 % were positive for M. tuberculosis and M. bovis respectively through PCR. Acid fast bacilli (AFB) have been instituted in 23 % sputum 08 % blood samples using Ziehl-Neelsen staining. On culturing of 11 % sputum and 09 % blood samples were found positive. The sensitivity and specificity of Duplex PCR was found statistically significant in comparison to conventional methods for the differential diagnosis of tuberculosis caused by M. tuberculosis and M. bovis. Therefore Duplex PCR is a better choice of diagnostic test in the clinical setups where clinical urgencies necessitate a reliable, sensitive and specific test with results in a short time period.