OBJECTIVES: To assess the mutation effects of ethambutol (EMB) resistance-associated genes, including embCAB operon, ubiA, embR, and aftA, on the EMB resistance levels among multidrug-resistant tuberculosis (MDR-TB) isolates from China. METHODS: A total of 159 MDR-TB from China had their EMB MICs quantified, and the sequences of the four ethambutol resistance-associated regions were analyzed. A multivariate regression model was established to evaluate the effects of mutations on EMB resistance. RESULTS: Our results showed that overall 95.6% (109/114 isolates) of EMB-resistant isolates harbored at least one mutation within the regions associated with EMB resistance. Most mutations were in embB, particularly in the embB300-500, and the embC-embA intergenic regions. Mutations in other genes were seldom seen and mainly occurred along with mutations in the embB or the embC-embA among the EMB-resistant isolates. DNA sequencing of the embB300-500 and the embC-embA was the most effective approach for detecting EMB resistance, with an accuracy of 91.2%. Nevertheless, some EMB-susceptible isolates still had a single mutation in the gene related to EMB resistance. Moreover, there was a significant correlation between EMB high-level resistance and multiple mutations. CONCLUSION: Distinct individual mutations, as well as multiple concurrent mutations, within EMB resistance-associated genes, contributed to variable levels of EMB resistance. These results have broadened our understanding of the molecular characteristics of EMB resistance in China.