This study aimed to investigate the prevalence of resistance to second-line antituberculosis (anti-TB) drugs and its association with resistance-related mutations in Mycobacterium tuberculosis isolated in China. In the present study, we collected 380 isolates from a population-based study in China and tested the drug susceptibility to first- and selected second-line drugs. These results were compared with polymorphisms in the DNA sequences of genes associated with drug resistance and MIC values of the studied second-line drugs. Of 43 multidrug-resistant M. tuberculosis isolates, 13 showed resistance to fluoroquinolones or injectable second-line drugs (preextensively drug-resistant TB [pre-XDR-TB]), and 4 were resistant to both and thus defined as extensively drug-resistant TB (XDR-TB). Age and previous TB therapy, including use of second-line drugs, were two independent factors associated with increased resistance to both first- and second-line drugs. Molecular analysis identified the most frequent mutations in the resistance-associated genes: D94G in gyrA (29.1%) and A1401G in rrs (30.8%). Meanwhile, all 4 XDR-TB isolates had a mutation in gyrA, and 3 of them carried the A1401G mutation in rrs. Mutations in gyrA and rrs were associated with high-level resistance to fluoroquinolones and the second-line injectable drugs. In addition to the identification of resistance-associated mutations and development of a rapid molecular test to diagnose the second-line drug resistance, it should be a priority to strictly regulate the administration of second-line drugs to maintain their efficacy to treat multidrug-resistant TB.