Non-tuberculous mycobacteria (NTM) are commonly encountered environmental bacteria, and most of them are associated with lung diseases. Diagnosis of infections caused by NTM is based on clinical, radiological and microbiological findings. The aim of this study was to investigate the distribution of non-tuberculous mycobacterial species isolated from clinical specimens as etiologic agents. The NTM strains isolated from clinical specimens in National Tuberculosis Reference Laboratory (NTRL), together with the strains that were sent to NTRL for the advanced identification of non-tuberculous mycobacterial species that have clinical or microbiological significance, were analysed retrospectively. The strains belonged to January 2009 - December 2010 period. If the same NTM type was isolated more than once in the clinical specimens of a patient, then it was defined microbiologically as a causative agent. Identification of mycobacteria species was performed by using a commercial line-probe assay (GenoType Mycobacterium CM/AS; Hain Lifescience, Germany). In our study, pulmonary and non-pulmonary samples obtained from 206 patients yielded mycobacterial growth in their cultures, and of them 24 (11.7%) were identified as NTM. On the other hand, 51 of the 101 samples sent to NTRL for identification were confirmed as NTM. Of the patients who were found to be infected with NTM (n= 75), 59 (78.7%) were male and the mean age was 50.9 ± 18.8 years. The most frequently identified NTM species was M.fortuitum (33.3%, n= 25), followed by M.abscessus (18.7%, n= 14), M.gordonae (10.7%, n= 8) and M.avium (%8; n= 6). The other types of NTM species identified in our laboratory were M.chelonae (n= 3), M.intracellulare (n= 3), M.kansasii (n= 3), M.peregrinum (n= 2), M.scrofulaceum (n= 2), M.szulgai (n= 2), M.celatum (n= 1), M.haemophilum (n= 1), M.smegmatis (n= 1) and M.xenopi (n= 1). Rapidly growing NTM species (M.fortuitum and M.abscessus) were the most frequent (52%) species isolated in our laboratory as the cause of non-tuberculous mycobacterial infection. Interestingly, the majority of M.fortuitum isolates (n= 21) which was the most common species identified in our laboratory, were those received from the peripheral laboratories. The most common species identified in our laboratory were rapidly growing NTM, however the countrywide distribution of the NTM species was found different than previously reported. In conclusion, further investigation of the non-tuberculous mycobacteria profile in adjunct with epidemiological data seems to be essential in our country.