Temporal changes in epidemiology of non-tuberculous mycobacteria (NTM) infections have hardly been explored. Frequency of isolation of different NTM species varies geographically. We aimed to determine whether the distribution of Mycobacteria species isolated from respiratory specimens had changed from 1996 to 2003. We analysed data on 484 patient mycobacterial isolates from 23,483 respiratory specimens submitted to the Laboratory of Microbiology in a tertiary medical centre. The proportion of patients with Mycobacterium tuberculosis isolates decreased from 44.6% in period I (1996–1999) to 20.6% in period II (2000–2003). Among the NTM, the proportion of some species increased significantly (Mycobacterium simiae: 8.4% to 31.6%; Mycobacterium fortuitum 12% to 20%; and Mycobacterium chelonae 4.8% to 11.3%), while others decreased (Mycobacterium avium complex 31.3% to 17.3%; Mycobacterium kansasii 28.9% to 7.5%; and Mycobacterium haemophilum 1.2% to 0%). These findings disclose major temporal changes in the distribution of mycobacterial species in respiratory specimens with an impressive emergence and takeover of M. simiae.