In this study, a total of 120 mycobacterial strains isolated from clinical specimens in Hacettepe University Hospital Clinical Pathology Laboratories were evaluated by polymerase chain reaction-restriction enzyme analysis (PRA), which analyses the common mycobacterial heat shock protein gene (hsp65). 95 of 120 strains (79.1%) were identified as Mycobacterium tuberculosis and 25 (20.8%) were identified as non-tuberculous mycobacteria (NTM). M. gordonae I and IV were the most common NTM species (3.3% each) followed by M. chelonae (2.5%). Other NTM species isolated were M. gordonae III, M. avium, M. peregrinum (1.6%), M. fortuitum, M. flavescens, M. malmoense and M. mucogenicum (0.8%). Four isolates had PRA patterns that did not match any patterns previously described. The patients who had NTM had underlying diseases; the most frequent clinical diagnosis among these was chronic obstructive pulmonary disease (COPD) and chronic renal failure. AIDS and pulmonary carcinoma were the other underlying diseases detected.