Recognition of differences, particularly in color and other growth characteristics, between Mycobacterium tuberculosis and other related organisms has led to the identification of the nontuberculosis mycobacteria (NTM). The Runyon Classification, based largely on the presence (or absence) of pigment and rate of growth, was a convenient system for classifying these organisms. Subsequent biochemical analysis has facilitated more precise speciation. However, many of these tests are complex and time consuming and beyond the capacity of most laboratories other than reference centers. The need for accurate identification has become clear because of differences in pathogenicity and treatment between various species and even subspecies of NTM. The recent appearance of rapid diagnostic tests and highly specific genetic identification systems have created new opportunities and challenges with respect to the identification of NTM. This article provides a historic context for the microbiologic study of the organisms, describes key features of the most commonly encountered NTM, notes several important new technical innovations in this field, and considers common clinical issues in relation to the microbiology of NTM.