Nontuberculous mycobacteria (NTM) are ubiquitous in hot and cold water distribution systems. With molecular typing methods it was shown that water can be the source of colonization and infection with NTM. The aim of our study was the investigation of NTM in hot and cold water samples taken from various departments of two hospitals in Istanbul, Turkey. Totally, 160 water samples were examined. The temperature, pH, and free chlorine levels of water samples were measured between 10–41 °C, 6.78–7.98 and <0.3–0.5 mg/L, respectively. NTM were detected in 33 (20.6%) samples. Totally 20 (60.6%), 10 (30.3%) and 3 (9.1%) isolates were identified as Mycobacterium lentiflavum, Mycobacterium gordonae, and Mycobacterium peregrinum, respectively. M. lentiflavum, which was the most frequently isolated NTM, is characterized by multiple resistance to antimycobacterial drugs. Although no infections with this mycobacterium were reported from our country so far, preventive measures may be considered in patients under immunosuppression. Because no significant correlations were found among the presence of NTM or species distribution and water temperature, pH or free chlorine levels, other factors need to be investigated.