Mycobacteria are subdivided into three groups: the Mycobacterium tuberculosis complex, the non-tuberculous mycobacteria called NTM or MOTT (Mycobacteria Other Than Tuberculosis) and Mycobacterium leprae. Over the past few decades, the incidence of infections caused by NTM has increased world wide. The differentiation of Mycobacterium tuberculosis complex from NTM is of primary importance for infection control and choice of antimicrobial therapy. However, there is so far no report in Bangladesh about the detection of NTM and hence differentiation of MTB and NTM. Neither acid-fast bacilli (AFB) staining nor histopathology can discriminate MTB and NTM. In order to detect and differentiate Mycobacterium tuberculosis complex and NTM we used commercially available LyteStar TB/NTM Real Time PCR kit (Altona Diagnostics, Germany) and analyzed 782 clinical specimens from tuberculosis suspected patients. We have found 49 MTB and 74 NTM positive samples from variety of clinical specimens such as sputum, bronchial lavages, body fluids, tissues, needle aspirates and swabs. Many of the PCR positive specimens were AFB negative on direct microscopic examination thus, indicating strong sensitivity of PCR than AFB staining. This is the first report in the country about detection of NTM and it warrants further elaborate investigation. Moreover, our results showed that multiplex real-time PCR assay is an effective sensitive tool for the rapid identification and differentiation of MTB and NTM directly from clinical specimens.