Diagnosis of infection due to nontuberculous mycobacteria is not easy, as it must be distinguished from colonization or contamination by other nontuberculous mycobacteria. Molecular methods offer many advantages over conventional methods of identification. The results are obtained rapidly, are reliable and reproducible, and even mixed or contaminated cultures can be examined. This review highlights the recent advances in molecular techniques for identification of nontuberculous mycobacteria.