Objective To analyze the drug resistance of sputum culture positive strains,and provide the clinical reference for tuberculosis prevention and control in Huaibei city. Methods A total of 302 positive strains of sputum culture were identified by drug sensitivity test which included first-line and second-line clinical drugs. 13 kinds of antituberculous drugs were identified. Results A total of 297 strains of mycobacterium tuberculosis and 5 non-tuberculous mycobacterium were isolated. There were 217 susceptible strains, accounting for 73.06%. There were 80 drug-resistant strains, and the resistance rate was 26.94%. There were 30 multidrug-resistant strains, and the resistance rate was 10.10%. There were 24 multi drug resistant strains, and the resistance rate was 8.08%. There were 8 single drug resistant strains, and the drug resistance rate was 8.42%. The drug resistance rate of newly treated patients was 22.39%, the drug resistance rate of re-treated patients was 36.46% and the difference was statistically significant. (χ²=6.54,P＜0. 05). Conclusion The proportion of drug resistant mycobacterium tuberculosis in Huaibei city was high, so we should pay more attention to the strategy of tuberculosis prevention and control.