Mycobacteria are the leading cause of death from infectious disease worldwide and limitations in current diagnostics are hampering control efforts. In recent years, the use of small volatile molecules as diagnostic biomarkers for mycobacteria has shown promise for use in the rapid analysis of in vitro cultures as well as ex vivo diagnosis using breath or sputum. In this study, 18 strains from four mycobacteria species (Mycobacterium avium, M. bovis BCG, M. intracellulare and M. xenopi) were analyzed for the first time using two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOFMS). This study represents the first time volatile molecules associated with M. intracellulare and M. xenopi have ever been reported. A total of 217 chromatographic features were identified and 58 features were selected that discriminate between these four species. Putative identifications are provided for 17 of the 58 discriminatory features, three of which have been reported previously in mycobacteria. The identification of mycobacteria-associated volatile biomarker suites could reduce the time-to-diagnosis for mycobacterial infections, either from in vitro cultures prior to the visualization of colonies or directly from ex vivo specimens, thereby shortening the empiric treatment window and potentially improving outcomes.