Novel tools are urgently needed for the rapid, reliable detection of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. To develop such tools, we need information about the frequency and distribution of the mycobacterial mutations and genotypes that are associated with phenotypic drug resistance. In a population-based study, we sequenced specific genes of M. tuberculosis that were associated with resistance to rifampin and isoniazid in 242 phenotypically MDR isolates and 50 phenotypically pan-susceptible isolates from tuberculosis (TB) cases in Shanghai, China. We estimated the sensitivity and specificity of the mutations, using the results of conventional, culture-based phenotypic drug susceptibility testing as the standard. We detected mutations within the 81-bp core region of rpoB in 96.3% of phenotypically MDR isolates. Mutations in two structural genes (katG and inhA) and two regulatory regions (the promoter of mabA-inhA and the intergenic region of oxyR-ahpC) were found in 89.3% of the MDR isolates. In total, 88.0% (213/242 strains) of the phenotypic MDR strains were confirmed by mutations in the sequenced regions. Mutations in embB306 were also considered a marker for MDR and significantly increased the sensitivity of the approach. Based on our findings, an approach that prospectively screens for mutations in 11 sites of the M. tuberculosis genome (rpoB531, rpoB526, rpoB516, rpoB533, and rpoB513, katG315, inhA-15, ahpC-10, ahpC-6, and ahpC-12, and embB306) could detect 86.8% of MDR strains in Shanghai. This study lays the foundation for the development of a rapid, reliable molecular genetic test to detect MDR strains of M. tuberculosis in China.Multidrug-resistant (MDR) tuberculosis (TB), defined as resistance to at least rifampin (RIF) and isoniazid (INH), and extensively drug-resistant (XDR) TB, defined as additional resistance to any fluoroquinolone and one injectable secondline drug, are among the most serious health threats of the 21st century. The epidemic of MDR TB is especially severe in China, a nation with the world's second largest number of TB cases and the largest number of MDR TB cases (39). A recent study reported that 9.3% of all TB cases in China are MDR, almost twice the worldwide MDR prevalence (4.8%) (14). While a lot of attention has been focused on acquired drug resistance among TB patients who receive an inadequate treatment regimen or who cannot adhere to their treatment regimen, several studies also showed that a large number of MDR TB cases are likely caused by transmission of MDR strains of Mycobacterium tuberculosis (2, 19). Therefore, there is an urgent need for new tools and approaches that will provide a rapid, reliable, and cost-effective diagnosis of MDR TB, particularly in resource-limited settings. This will help to prevent transmission of MDR strains and to optimize treatment regimens for MDR cases.Drug susceptibility testing by the conventional solid medium culture method is highly sensitive and specific but extremely ...