The goal of this research was detection of the drug-resistance level of Mycobacterium tuberculosis infection in the Odesa region of Southwest Ukraine, investigation of the level of mutation in katG and rpoB genes for M. tuberculosis with polymerase chain reaction (PCR), and spread of these mutations in different groups of patients with tuberculosis. An extremely high level of primary and acquired resistance of M. tuberculosis to first-line antituberculosis drugs has been found in the Southwest region of Ukraine. The PCR method has proved to have high sensitivity in the detection of mutations in katG and rpoB genes. The data showed significant spreading of M. tuberculosis strains with mutations in katG and rpoB genes in penitentiaries and an increased level of these mutations during tuberculosis treatment. The presence of mutations in rpoB and katG genes was associated with a more severe course of tuberculosis, increased risk of treatment default, persistence of positive smears on microscopy at discharge, and poor closing of tuberculous cavities. Extremely high level of mutations in the rpoB and katG genes of M. tuberculosis was observed in Beijing family strains. Our findings support the capability of the PCR method to detect M. tuberculosis that is resistant to drugs such as isoniazid and rifampicin.