Patients infected with Non-tuberculous mycobacteria (NTM) usually do not respond to conventional anti-tubercular treatment and are misdiagnosed as infection with multi-drug resistant strains of mycobacterium tuberculosis (M.tb) due to lack of correct species identification, particularly in the developing countries like India. One of the challenges faced by clinicians in the treatment of tuberculosis is the absence of an easy, reliable and rapid identification tool that can accurately differentiate disease caused by M.tb complex from NTM. Keeping this in consideration, the performance of species specific nucleic acid probe i.e. Accuprobe was assessed and compared with conventional niacin production, nitrate reductase assay techniques for identification of M.tb complex in 80 mycobacterial isolates obtained from different extra-pulmonary sites. Accuprobe identified 62 isolates (77.5%) as M. tuberculosis complex and remaining 18 isolates (22.5%) as NTM whereas 64 isolates (80%) were identified as M.tb and rest 16 (20%) were interpreted as NTM by conventional biochemical techniques. The overall agreement between both techniques was 96.9% The sensitivity, specificity, positive predictive value(PPV) and negative predictive value (NPV) shown by accuprobe were 96.9%, 100%, 96.9%, and 88.9% respectively. Thus, accuprobe has showed impressive sensitivity and specificity giving results in < 3 hrs from culture-positive isolates and have sure edge over conventional biochemical methods which are, nonetheless, labour intensive and cumbersome to perform thus delaying prompt mycobacterial identification.