Classical methods for identification of Mycobacterium species rely on morphology and biochemical profiles. Speciation of a Mycobacterium isolate using these standard methods is a lengthy process based on subjective data interpretation. In this study, Mycobacterium species were characterized by utilizing matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This technology is designed to provide a characteristic mass spectral fingerprint based on desorbed ions from the cell surface. Thirty-seven strains were analyzed; these represented thirteen species and five subspecies that included the Mycobacterium tuberculosis complex and the M. avium-M. intracellulare complex, as well as rapid- and slow-growing mycobacteria. All 37 strains were analyzed in triplicate, and a database was generated. This method produced species-specific patterns for all but 1 of the 37 isolates and provided reliable differentiation at the strain level. The data suggest that whole-cell MALDI-TOF MS has potential as a rapid and reproducible method for the identification and characterization of Mycobacterium species.