The aim of this study was the identification of atypical mycobacteria isolated from various samples of patients prediagnosed as tuberculosis between November 2004 and June 2006 by a commercial polymerase chain reaction (PCR) based reverse hybridization kit (INNO-LiPA Mycobacteria v2, Innogenetics NV, Belgium). A total of 21,060 samples obtained from 9660 patients were included to the study. After decontamination and homogenization processes, the samples were cultivated in automated MGIT Bactec 960 system and the diagnosis of atypical mycobacteria was performed in 4532 (21.5%) culture positive samples with NAP test by using Bactec 460 TB system. After DNA isolation, PCR method was performed by using the primers specific for mycobacterial 16S-23S spacer region. PCR products were then hybridized with the probes specific for Mycobacterium species on nitrocellulose strips according to the recommendations of the manufacturer and evaluated. Additionally, two different versions of another commercial Line Probe Assay (LiPA) kit [GenoType Mycobacterium and GenoType Mycobacterium AS (Additional Species), Hain Lifescience, Germany] were used for the detection of unidentified mycobacterial strains. In our study period, 10 different Mycobacterium species were identified from 44 (1%) respiratory tract samples (sputum, bronchial aspiration fluid, bronchoalveolar lavage) belonging to 30 patients. While repeated atypical mycobacterial growth was found in 13 patients on different days, 17 patients showed atypical mycobacterial growth only in one sample or in separate multiple samples taken within the same day. The species distribution among patients were as follows; M. fortuitum-M. peregrinum complex (n=5), M. intracellulare (n=4), M. avium complex (n=4), M. chelonae complex (n=4), M. gordonae (n=4), M. kansasii (n=3), M. szulgailintermedium (n=2), M. simiae (n=1) and M. scrofulaceum (n=1). Two of four samples which were unidentified by INNO-LiPA and GenoType MTBC were identified as M. szulgai/intermedium by GenoType Mycobacterium AS and the other two were found as unidentified atypical mycobacteria (Mycobacterium spp.). As a result, the frequency of atypical mycobacteria isolated in our hospital was thought to be low, however, species-level identification might be useful for the planning of therapy in such patients. In addition, after NAP test, INNO-LiPA and GenoType Mycobacterium were useful tests in microbiological identification of atypical mycobacteria, and GenoType Mycobacterium AS test could be applied in mycobacterial strains which were not identified by the former assays.