We have conducted a multicenter study of the diagnostic accuracy of the MTBDRplus 2.0 assay in compared with conventional and molecular reference standard in four tuberculosis (TB)-specialized hospitals of China. A total of 5038 patients were enrolled in this study. The overall sensitivity of the assay for the diagnosis of TB was 92.7% [1723/1858, 95% confidence interval (95% CI): 91.5-93.9]. In smear-positive/culture-positive cases the sensitivity was 97.7% (995/1018, 95% CI: 96.6-98.6), whereas in smear-negative/culture-positive cases it was 86.7% (728/840, 95% CI: 84.2-88.9). The agreement rate between MTBDRplus 2.0 and Xpert MTB/RIF was 97.7% (1015/1039, 95% CI: 96.6-98.5) for smear-positive cases and 97.0% (3682/3794, 95% CI: 96.5-97.6) for smear-negative cases. As compared with phenotypic drug susceptibility testing, the MTBDRplus 2.0 correctly identified 298 of 315 patients (94.6%, 95% CI: 91.5-96.8) with rifampicin-resistance. As noted previously, isoniazid resistance is associated with many different mutations and consequently the sensitivity compared to phenotypic testing was lower (81.0%, 95% CI: 76.8-84.7). In conclusion, this assay is a rapid, accurate test in terms of increased sensitivity for detecting smear-negative TB patients, as well as an alternative for detecting both RIF and INH resistance in persons with presumptive TB, whereas the absence of a mutation in the specimens must be interpreted cautiously.