Worldwide, tuberculosis (TB) remains one of the leading infectious diseases, accounting for nearly 3 million deaths and more than 8 million new cases annually. Among them are 3.2 % multidrug-resistant strains of Mycobacterium tuberculosis. In the control of the spread of TB clinical mycobacteriology laboratories play an essential role providing the clinicians with timely detection, isolation, identification, and drug susceptibility testing results for M. tuberculosis. In the past twenty years numerous molecular techniques have been introduced to comply with the obvious needs for a reliable and rapid diagnosis of the disease. Nucleic acid amplification-based assays, in particular, the polymerase chain reaction (PCR), allow (i) direct detection of M. tuberculosis complex in clinical specimens; (ii) identification of mycobacteria; (iii) detection of resistance of M. tuberculosis to antimicrobial agents; and (iv) DNA typing to answer questions such as reactivation of disease or exogenous reinfection, and to track transmission and internal laboratory contaminations.