Multidrug resistant (MDR) tuberculosis has emerged as a major public health problem worldwide. However, knowledge of the regional complete susceptibility to second-line drugs is rare, which impedes development of a global strategy of tuberculosis control. Presently, we determined the susceptibility of 125 isolates from southern Taiwan to 5 first-line and 12 second-line antituberculosis drugs. Except for a lower resistance to ethionamide (9.6% of isolates), more than 20% of the isolates were resistant to second-line drugs (kanamycin, 29.6%; p-aminosalicyclic acid, 71.2%; cycloserine, 24.8%; capreomycin, 24.8%; and ofloxacin, 28.5%). Twenty-two (17.6%) MDR strains displayed higher resistance to second-line antituberculous agents, compared with non-MDR strains, with markedly higher resistance rates evident for ethambutol, pyrazinamide, streptomycin, kanamycin, and ofloxacin. For clofazimine, amikacin, clarithromycin, ciprofloxacin, and amoxicillin/clavulanate, the MIC(90 )of the MDR isolates all exceeded those of non-MDR isolates. Moreover, four extensively drug resistant (XDR) strains first found in Taiwan, accounted for 3.2% of all isolates. The high resistance to the second-line drugs, especially among MDR strains, stresses the importance of proper treatment in Taiwan and threatens the global control of tuberculosis.