Abstract: Objective To investigate the association between embB mutation of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) and its ethambutol(EMB) resistance. Methods Proportion method was used to detect the EMB resistance of 84 strains of MDR-TB and gene sequencing was conducted to detect the mutation of embB gene. The association between embB mutation and the ethambutol resistance was also analyzed with chi-square test. Results Among the 84 strains of MDR-TB, 43 were resistant to EMB(51.2%), 41 were sensitive to EMB(48.8%). embB mutation occurred in 57 MDR-TB strains(67.9%) and in 40 out of 43 EMB resistant strains(90.3%). Among the 41 EMB sensitive strains, embB mutation was detected in 17 strains(41.5%). The mutation rate of embB was much higher in EMB resistant strains than in EMB sensitive strains. embB306 was the major mutation loci, and the mutation occurred at this loci in EMB resistant strains was much higher in EMB sensitive strains.(2=12.37, P=0.00). The sensitivity, specificity and accuracy of EMB susceptibility test in embB and at embB306 were 93.0% and 65.1%, 58.5% and 73.2%, and 76.2% and 69.0% respectively. Conclusion EMB resistance was associated with embB and mutation at embB306.