The genus Mycobacterium, belonging to the Actinobacteria, is closely related to Corynebacterium, Nocardia, Propionibacterium, and Rhodococcus. It consists of over 130 species, of which some cause serious infectious diseases such as tuberculosis and leprosy in animals and humans. Mycobacteria are globally spread, typically living in a variety of environments, including water and food sources. They are aerobic and nonmotile bacteria that are characterized by their acid-alcohol fast staining properties. All mycobacterial species share a characteristic cell wall, which is hydrophobic, waxy, and rich in mycolic acids. A natural division is drawn between slow and rapid growing species. In this chapter, we describe the current diagnostic microbiology methods applied for isolation and identification of Mycobacteria. Applications of immunological and molecular tests for the diagnosis of M. tuberculosis infection are discussed. During the last two decades molecular epidemiology techniques have been developed and widely applied. We describe applications of several standardized tests such as spoligotyping, MIRU-VNTR, IS6110-RFLP, and SNP typing. Mycobacterium tuberculosis strain molecular genotypes are stored in databases. The existing molecular epidemiology databases play an important role in improving the investigations of the global evolution of M. tuberculosis worldwide.