We have developed and evaluated a semiautomated assay for detection of nontuberculous mycobacteria (NTM) from clinical samples based on the COBAS Amplicor M. tuberculosis test (Roche Diagnostics, Switzerland). A capture probe, specific for mycobacteria at the genus level, was linked to magnetic beads and used for the detection of amplification products obtained by the COBAS Amplicor M. tuberculosis assay. We demonstrate that the analytical sensitivity of the genus assay is similar to that of COBAS Amplicor M. tuberculosis detection. 416 clinical specimens were evaluated for the presence of NTM DNA. Sensitivity for smear positive and smear negative specimens was found to be 100 % and 47.9 %, respectively. Specificity was 97.7 %, the positive predictive value (PPV) 84.6 % and the negative predictive value (NPV) 93.1 %. The genus assay is easy to perform, produces reliable results and was found to be a valuable diagnostic tool for rapid diagnosis of infections with NTM. The genus assay has the potential to detect NTMs not routinely recovered by culture and to discover new mycobacterial species.