ABSTRACT Mycobacterium tuberculosis complex, Mycobacterium avium , and many other nontuberculous mycobacteria are worldwide distributed microorganisms of major medical and veterinary importance. Considering the growing epidemiologic significance of wildlife-livestock-human interrelation, developing rapid detection tools of high specificity and sensitivity is vital to assess their presence and accelerate the process of diagnosing mycobacteriosis. Here we describe the development and evaluation of a novel tetraplex real-time PCR for simultaneous detection of Mycobacterium genus, M. avium subspecies, and M. tuberculosis complex in an internally monitored single assay. The method was evaluated using DNA from mycobacterial ( n = 38) and nonmycobacterial ( n = 28) strains, tissues spiked with different CFU amounts of three mycobacterial species ( n = 57), archival clinical samples ( n = 233), and strains isolated from various hosts ( n = 147). The minimum detectable DNA amount per reaction was 50 fg for M. bovis BCG and M. kansasii and 5 fg for M. avium subsp. hominissuis . When spiked samples were analyzed, the method consistently detected as few as 100 to 1,000 mycobacterial CFU per gram. The sensitivity and specificity values for the panel of clinical samples were 97.5 and 100% using a verified culture-based method as the reference method. The assays performed on clinical isolates confirmed these results. This PCR was able to identify M. avium and M. tuberculosis complex in the same sample in one reaction. In conclusion, the tetraplex real-time PCR we designed represents a highly specific and sensitive tool for the detection and identification of mycobacteria in routine laboratory diagnosis with potential additional uses.