Background & Objective: Nontuberculous mycobacteria (NTM) especially rapid growing mycobacteria (RGM) are opportunistic environmental pathogens and their role in human disease is increasingly recognized. In addition, several studies indicated that humans can be infected by NTM from various environmental sources especially soil and water. Identification of rapid growing mycobacteria is important for both clinical and epidemiological studies because of their worldwide propagation. Incidence of rapid growing mycobacteria from water and soil in different regions of Robat Karim was studied using PCR-RFLP method and targeting hsp65 gene and 16S-23SrRNA. Methods: In this study a total number of 454 water and soil samples were collected from different areas of Robat Karim. After decontamination of isolates by petroff method, the sediment obtained was cultured onto Lowenstein Jensen media and Ziehl Neelsen staining was performed for microscopic observations. After DNA extraction, hsp65 gene amplified by PCR-RFLP and then PCR products were digested by two restriction enzymes, BsteII and HaeIII, prior to undergoing polyacrilamid gel electrophoresis (PAGE). 16S-23S rRNA gene was also amplified and then PCR products were digested by restriction enzymes of HaeIII and CfoI and analyzed using PAGE. Results: Based on the culture and microscopic observations, 49 (21.5%) out of 227 soil samples and 144 (63%) out of 227 water samples were positive regarding mycobacterium. 7 soil samples and 29 water samples were contained rapid growing mycobacteria based on PCR-RFLP method. The results obtained by molecular method matched completely with those from culture method. Therefore, identification of Rapid Growing Mycobacterium has a sensitivity of 100% and mycobacterium fortuitum was the predominant isolated rapid growing mycobacteria in water and soil samples with a frequency of 27.8%. Conclusion: Mycobacterium fortuitum was the most dominant rapid growing mycobacterium isolated from soil and water samples in Robat Karim area.