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The number of immunocompromised patients has increased significantly over the past two decades, introducing previously unrecognized organisms or those considered clinically insignificant into the medical laboratory. The further introduction of sensitive methods for isolation of mycobacteria, especially of the non-tuberculous mycobacteria (NTM), together with more accurate methods for their identification have caused the recognition of a large number of new species in association with human disease. Additionally, molecular based techniques permit separation of significantly variant mycobacteria which had previously been lumped into single species. The vaccine strain of Mycobacterium bovis, bacille Calmette–Guérin (BCG), a member of the M. tuberculosis complex, is more commonly becoming a pathogen in certain patients and may be under-diagnosed. A larger number of NTM are also being identified and some are periodically implicated in disease processes. More recently, organisms such as M. asiaticum, M. branderi, M. celatum, M. interjectum, M. intermedium, M. mucogenicum, M. neoaurum, M. phlei, M. shimodei, M. thermoresistibile and M. triplex have been described in the clinical laboratory and are included in this review. Several problems still exist, including: (i) accurate and timely identification of isolates; (ii) standardization and clinical evaluation of susceptibility studies; and (iii) capability to differentiate significant from insignificant isolates.