Rifabutin is a spiro-piperidyl-rifamycin derived from rifamycin-S. This study was planned to determine the minimal inhibitory concentrations (MICs) of rifabutin and rifampicin against Mycobacterium tuberculosis strains and to investigate cross resistance between two drugs. The agar dilution method was performed for detection of MICs of rifabutin and rifampicin for 112 M. tuberculosis strains obtained from patients with active pulmonary tuberculosis. Cross resistance between rifampicin and rifabutin was detected in 73.1%. Rifabutin MICs for rifampicin resistant strains were three- to four-fold higher than rifabutin-susceptible and/or -resistant strains' MIC values for rifampicin. Despite having 73% cross-resistance with rifampicin in M. tuberculosis isolates, it was concluded that rifabutin might be a valid component of combination therapy in rifampicin resistant tuberculosis cases when the isolate is tested and detected susceptible to rifabutin.