Aims: Polymerase chain reaction ( PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management. Methods and Results: Two hundred and thirty-five samples from 145 clinically suspected cases of tuberculosis were processed for the detection of Mycobacterial infections by ZN (Ziehl Neelsen) smear examination, L-J & BACTEC (TM) MGIT-960 culture and multiplex PCR tests. The multiplex PCR comprised of genus-specific primers targeting hsp65 gene, Mycobacterium tuberculosis complex-specific primer targeting cfp10 (Rv3875, esxB) region and Mycobacterium avium complex-specific primer pairs targeting 16S-23S Internal Transcribed Spacer sequences. The multiplex PCR developed had an analytical sensitivity of 10 fg (3-4 cells) of mycobacterial DNA. The multiplex PCR test showed the highest (77.24%) detection rate, while ZN smear examination had the lowest (20%) detection rate, which was bettered by L-J culture (34.4%) and BACTEC (TM) MGIT-960 culture (50.34%) methods. The mean isolation time for M. tuberculosis was 19.03 days in L-J culture and 8.7 days in BACTEC (TM) MGIT-960 culture. Using the multiplex PCR, we could establish M. tuberculosis + M. avium co-infection in 1.3% HIV-negative and 2.9% HIV-positive patients. The multiplex PCR was also highly useful in diagnosing mycobacteraemia in 38.09% HIV-positive and 15.38% HIV-negative cases. Conclusions: The developed in-house multiplex PCR could identify and differentiate the M. tuberculosis and M. avium complexes from other Mycobacterial species directly from clinical specimens. Significance and Impact of the Study: The triplex PCR developed by us could be used to detect and differentiate M. tuberculosis, M. avium and other mycobacteria in a single reaction tube.