Tuberculosis is one of the leading infectious diseases in the world and is responsible for more than 2 million deaths and 8 million new cases annually. Because of the slow growth rate of the causative agent Mycobacterium tuberculosis, isolation, identification, and drug susceptibility testing of this organism and other clinically important mycobacteria can take several weeks or longer. During the past several years, many molecular methods have been developed for direct detection, species identification, and drug susceptibility testing of mycobacteria. These methods can potentially reduce the diagnostic time from weeks to days. Currently, two nucleic acid amplification methods, the Enhanced Mycobacterium tuberculosis Direct Test (Gen-Probe) and the Amplicor Mycobacterium tuberculosis Test (Roche Diagnostic Systems), have been approved by the Food and Drug Administration for direct detection of M. tuberculosis from clinical specimens. PCR-based sequencing has become commonly used to identify many mycobacterial species. DNA probes have been widely used for species determination of the most commonly encountered mycobacteria. High-density oligonucleotide arrays (DNA microarrays) also have been applied to simultaneous species identification and detection of mutations that confer rifampin resistance in mycobacteria.