Drinking water has been identified as an important source of various infectious diseases including opportunistic infections caused by Non-Tuberculous Mycobacteria (NTM) in humans. In this study the occurrence of mycobacterium species were examined in drinking water samples (n=198) collected from reverse osmosis water and tap water systems of hospital and household settings in Agra region of Uttar Pradesh using Ziehl-Neelsen staining and culture on Lowenstein–Jensen medium method. Acid-fast isolates were identified by PCR amplification and analysis of restriction endonuclease digestion fragments of the heat-shock protein 65 (hsp65) gene. Of the 198 drinking water samples, 22 (11.11%) samples were found positive for the presence of mycobacterium species. The presence of mycobacterium species was found higher in tap water (17.07%) as compare to reverse osmosis water (6.89%) systems and the difference was statically significant (χ2: 5.548, p-value: 0.018). The result of hsp65PCR revealed the presence of seventeen mycobacterium species predominated by M.chelonae, M.flavescenes and M. intracellulare in drinking water. Present study reported the occurrence, distribution and diversity of mycobacterium species in drinking water systems in Agra region of Uttar Pradesh and highlights the need of health policies aimed at mitigating the risk of NTM exposure through drinking water in India.