Objective To evaluate the application effect of broth microdilution (BMD) method in susceptibility testing of multidrug-resistant Mycobacterium tuberculosis (MDR-MTB). Methods The Roche's proportion method and BMD method were adopted in drug susceptibility testing on 108 MDR-MTB strains and 11 non-MDR-MTB strains in Hainan Province. Whole genome sequencing (WGS) was performed on strains with inconsistent results by the above two methods. Results The average time to acquire drug susceptibility testing results by Roche's proportional method and BMD method were 28.0 and 8.5 days, respectively. Roche's proportional method showed higher resistance rates to isoniazid (INH), rifampicin (RFP), ethambutol (EMB), kanamycin (KM), and capreomycin (CPM) than BMD method (all P < 0.001). BMD method showed higher resistance rates to protionamide (PTO) and para-aminosalicylic acid (PAS) than Roche's proportional method (both P < 0.001). Taking Roche's proportional method as the gold standard, the sensitivity and specificity of BMD method for testing drug resistance were 50.00%-100% and 95.69%-100%, respectively. Except EMB (87.39%) and INH (94.96%), the consistency rates of the BMD method in testing drug resistance of other drugs were all ≥95.00%. The overall consistency rate between Roche's proportional method and WGS was 76.19% (32/42), while the consistency rate between BMD method and WGS was 23.81% (10/42), difference was statistically significant (χ²=23.048, P < 0.001). 34 MTB strains showed inconsistent results by two drug susceptibility testing methods. Among the 26 MTB strains that were resis-tant in Roche's proportion method but sensitive in BMD method, 22 strains (84.62%) had mutations in relevant resistance genes. Among the 11 MTB strains that were sensitive in Roche's proportion method but resistant in BMD method, 5 strains (45.45%) had mutations in relevant resistance genes. Conclusion BMD method is an accurate and rapid MDR-MTB susceptibility testing method, but further improvement and optimization are still needed. Drug resistance is closely related to mutations in relevant resistance genes.