bThe Abbott RealTime MTB (RT MTB) assay is a new automated nucleic acid amplification test for the detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. In combination with the RealTime MTB INH/RIF (RT MTB INH/RIF) resistance assay, which can be applied to RT MTB-positive specimens as an add-on assay, the tests also indicate the genetic markers of resistance to isoniazid (INH) and rifampin (RIF). We aimed to evaluate the diagnostic sensitivity and specificity of RT MTB using different types of respiratory and extrapulmonary specimens and to compare performance characteristics directly with those of the FluoroType MTB assay. The resistance results obtained by RT MTB INH/RIF were compared to those from the GenoType MTBDRplus and from phenotypic drug susceptibility testing. A total of 715 clinical specimens were analyzed. Compared to culture, the overall sensitivity of RT MTB was 92.1%; the sensitivity rates for smear-positive and smear-negative samples were 100% and 76.2%, respectively. The sensitivities of smear-negative specimens were almost identical for respiratory (76.3%) and extrapulmonary (76%) specimens. Specificity rates were 100% and 95.8% for culturenegative specimens and those that grew nontuberculous mycobacteria, respectively. RT MTB INH/RIF was applied to 233 RT MTB-positive samples and identified resistance markers in 7.7% of samples. Agreement with phenotypic and genotypic drug susceptibility testing was 99.5%. In conclusion, RT MTB and RT MTB INH/RIF allow for the rapid and accurate diagnosis of tuberculosis (TB) in different types of specimens and reliably indicate resistance markers. The strengths of this system are the comparably high sensitivity with paucibacillary specimens, its ability to detect INH and RIF resistance, and its high-throughput capacities. R apid and accurate diagnosis of tuberculosis (TB) and fast detection of drug resistance are essential to ensure early initiation of appropriate antituberculotic treatment, adequately manage the disease, and control further transmission. Worldwide, one-third of all TB cases and almost three-quarters of the 480,000 cases of multidrug-resistant (MDR; defined as resistance toward rifampin [RIF] and isoniazid [INH]) TB are not reported, with the vast majority of them occurring in high-burden countries (1). Molecular tests are the most promising tools to close this diagnostic gap. Consequently, nucleic acid amplification tests (NAATs), such as PCR assays that allow for the fast and accurate detection of Mycobacterium tuberculosis complex (MTBC) DNA directly in clinical specimens, have become an indispensable tool in TB diagnostics over the last several decades. Most commercial tests show excellent specificity and sensitivity rates with smear-positive specimens while sensitivity rates range from 49% to 78% with smearnegative samples (2-7).Particularly in regions with high prevalences of MDR-TB, the molecular detection of genetic markers of resistance directly in the clinical specimen is playing a pivotal role in early not...