Mycobacterium is still is one of the major causes of mortality, since two million people die each year from this malady. Currently, there are over 170 recognized species of Mycobacterium, the only genus in the family Mycobacteriaceae. Organisms belonging to this genus are quite diverse with respect to their ability to cause disease in humans; some are strict pathogens, while others are opportunistic pathogens or nonpathogenic. Similar to other major groups of bacteria, the mycobacteria have undergone an extraordinary expansion in the number of different species over the last 2 decades, due in large part to the discriminatory power of gene sequencing, which phenotypic methods cannot achieve. This discriminatory power is such that phenotypic traits, i.e., biochemical and cultural characteristics, are no longer acceptable for the identification of mycobacteria. M. tuberculosis strains are reportedly more virulent than others, as defined by increased transmissibility as well as being associated with higher morbidity and mortality in infected individuals. As more clinical laboratories use molecular or other methods, such as mass spectrometry, for identification, our understanding of clinical significance will change and evolve as the number of case reports regarding the “new” species increase; it may well be that the role of many of the newly described mycobacterial species has been underestimated either because of misidentification or because the species were unrecognized. 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 the Mycobacterium species under investigation as well as on the research question.. Within this review, we summarize currently available molecular methods for strain typing of M. tuberculosis. For the various methods, technical practicalities as well as discriminatory power and accomplishments are reviewed.