The amplification of a fragment from hsp65 gene by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis with BstEII and HaeIII restriction enzymes has demonstrated to be very useful for identification of Non-Tuberculous Mycobacteria (NTM). The biochemical tests as well as the PCR-RFLP were carried out in 13 reference strains and 46 strains received in the laboratory. The results by biochemical tests were available in 4-6 weeks whereas the PCR-RFLP only required 48 hours. In both methods, Mycobacterium intracellulare, M. kansasii and M. fortuitum were the most frequently detected species. The PCR-RFLP method is fast, cheap and simple. Its application in Reference Laboratories could be very useful for diagnosis of NTM.