We performed the first evaluation study of the new FluoroType MTB assay, a HyBeacon based nucleic acid amplification test for the direct detection of Mycobacterium tuberculosis in respiratory tract specimens. Using mycobacterial culture as a reference, 661 specimens were tested. Sensitivity and specificity were 95.1% and 96.4% for all specimens, sensitivity was 100% for smear-positive and 84.6% for smear negative specimens, respectively. The FluoroType MTB assay showed fast and accurate results within 3 hours, including DNA extraction.