A total of 92 Mycobacterium tuberculosis isolates were collected from patients with pulmonary tuberculosis (TB) in the Zunyi region between 2011 and 2012. Collected isolates were used to determine antibiotic susceptibility patterns against 13 anti-TB drugs: 4 first-line and 9 second-line (ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, para-aminosalicylic acid, amikacin, capreomycin, kanamycin, and prothionamide) drugs. Results showed that among 57 new cases of TB only 66.7% were susceptible to all four first-line anti-TB drugs and 64.9% were susceptible to fluoroquinolones and second-line injectables; 10.5% of new and 22.9% of previously treated cases were multidrug-resistant TB (MDR-TB); and 1.8% of new and 2.9% of previously treated cases were extensively drug-resistant TB (XDR-TB). In addition, 14.3% of MDR-TB cases (2 out of 14) were XDR-TB, which is higher than the average numbers in China (about 8%) and in the world (9.6%). This study confirms that primary transmission of drug-resistant TB, including MDR/XDR-TB, is a real threat to achieving effective control of drug-resistant TB in the Guizhou Province and indicates the necessity to determine antibiotic susceptibility patterns in patients with TB to improve treatment outcomes.