In the epidemiology of tuberculosis (TB) and nontuberculous mycobacterial (NTM) diseases, as in all infectious diseases, the key issue is to define the source of infection and to disclose its routes of transmission and dissemination in the environment. For this to be accomplished, the ability of discerning and tracking individualMycobacteriumstrains is of critical importance. Molecular typing methods have greatly improved our understanding of the biology of mycobacteria and provide powerful tools to combat the diseases caused by these pathogens. The utility of various typing methods depends on theMycobacteriumspecies under investigation as well as on the research question. For tuberculosis, different methods have different roles in phylogenetic analyses and person-to-person transmission studies. In NTM diseases, most investigations involve the search for environmental sources or phylogenetic relationships. Here, too, the type of setting determines which methodology is most suitable. Within this review, we summarize currently available molecular methods for strain typing ofM. tuberculosisand some NTM species, most commonly associated with human disease. For the various methods, technical practicalities as well as discriminatory power and accomplishments are reviewed.