Tehran University of Medical Sciences Journal of Medical Bacteriology 2251-8649 13 2 2025 05 05 31 39 Paniz Mahmoudi Department of Microbiology, Faculty of Modern Sciences and Technologies, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. Kumarss Amini Department of Microbiology, Faculty of Sciences, Saveh Branch, Islamic Azad University, Saveh, Iran. Sarvenaz Falsafl Department of Microbiology, Faculty of Modern Sciences and Technologies, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. 2025 01 17 2025 02 06 Background:   Non-tuberculous mycobacteria (NTM) pose a growing public health concern, especially in regions with high prevalence rates. Iran, situated near high-burden countries like Afghanistan and Pakistan, is particularly vulnerable. This study aimed to investigate the molecular epidemiology of NTM in water samples from Farhikhtegan Hospital, Tehran. Methods:   A total of 70 water samples were collected from various hospital departments. After culture and phenotypic identification, 33 mycobacterial isolates were subjected to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the rpoB gene. Results:   Mycobacterium fortuitum type I was the most prevalent NTM species, accounting for 81% of the isolates. Mycobacterium kansasii type I and Mycobacterium gordonae type I followed, each comprising 6% of the isolates. Less common species included M. gordonae type II and Mycobacterium intracellulare. The PCR-RFLP method proved to be a sensitive and accurate tool for identifying NTM species. Conclusion:   This study provides valuable insights into the NTM profile in Farhikhtegan Hospital and underscores the importance of effective surveillance and control measures to mitigate the risk of NTM infections. https://jmb.tums.ac.ir/index.php/jmb/article/view/558