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Background and objective: Nontuberculosis mycobacteria (NTM) are free-living saprophytes that are found in water, soil, animals and dairy products. During recent years, advances in molecular biology improved the recovery and accuracy of NTM identification. In the present study, similar approaches were used to differentiate environmental NTM. For this reason, the prevalence of environmental NTM in soil and water in two densely populated areas of Tehran were investigated. Material and Methods: A total of 2,540 samples were collected from soil (n = 1237) and water (n = 1303) resources. For the soil sample: digestion and decontamination were performed using modified Engbaeks method, and for the water sample: decontamination followed with ethylpyridinium chloride (CPC; final concentration of 0.05%) and standard protocol. The phenotypic and genotyping tests were performed on all positive cultures. For molecular tests, both 16S-23S RNA and hsp65 genes spacer PCR-RFLP were used. Results: Of the 2,540 collected samples, 285 (12%) were positive for mycobacterium. One hundred and sixty-nine (169; 60%) belonged to a slow growing mycobacteria (SGM) and 116 (41%) were rapid growing mycobacteria (RGM). The most frequent NTM were identified as Mycobacterium farcinogenes (51/485; 10.5%) and Mycobacterium fortuitum (38/485; 7.8%). However, M. farcinogenes is found to be dominant in both studied areas, but the frequency of RGM was different. In Shahre-ray, Mycobacterium senegalense was identified as the most frequent RGM (22/65; 34%), while in Varamin, M. fortuitum (20/51; 40%) had the most frequency. Conclusion: The molecular genotyping methods facilitate the rapid identification of environmental NTM. As the higher prevalence of NTM within a region increases the risk of human infection, this study outlines the importance of further investigation.