Tuberculosis remains a leading cause of morbidity and mortality worldwide. A rapid and reliable diagnosis and discrimination from infections with nontuberculous mycobacteria (NTM) is critical. Frequently, formalin-fixed, paraffin-embedded (FFPE) tissues remain the only source for detection of micro-organisms in suspected cases of mycobacterial infection. Recently, numerous methods, including PCR assays, in situ hybridization and immunohistochemistry have been developed for detection of mycobacteria in FFPE samples. PCR-based assays are directed either against M.tbc.-specific sequences, such as IS6110, or amplify regions common to many mycobacterial species, e.g. the 65 kDa antigen, and then require sequencing or restriction fragment length polymorphism for species identification. Whereas the detection of DNA of M.tbc. in the correct setting is always of clinical relevance, the presence of various NTM species has to be interpreted with great caution due to their ubiquitous nature. However, the routine application of molecular tests has demonstrated that NTM infections are more common than previously thought, even in non-immunosuppressed hosts. The introduction of real-time PCR technology allows precise quantification of mycobacterial DNA and can be used for species identification through melting point analysis or appropriate DNA probes. Application of these assays originally developed for clinical microbiology offer a great opportunity for diagnostic improvement in molecular pathology as compared to qualitative PCR, mainly due to an increased specificity and a lower risk of contamination. Given the clinical impact of a positive molecular result for M. tbc., future efforts have to be aimed at standardization and quality control.