Kazakhstan is one of the 14 countries with a high rate of morbidity due to multidrug-resistant tuberculosis (MDR TB) in WHO European region. The aim of our study was to characterize mutations associated with drug resistance to rifampicin and isoniazid in Mycobacterium tuberculosis isolates from Kazakhstan. M. tuberculosis strains were isolated from TB patients in different regions of Kazakhstan. A drug susceptibility test was performed on Lowenstein-Jensen medium using the absolute concentration method. Sequencing analysis was performed of the rpoB rifampicin resistance-determining region and the katG gene, the oxyR-ahpC intergenic region, and the inhA promoter region in 259 MDR M. tuberculosis isolates, in 51 isoniazid-resistant isolates, and in 13 rifampicin-resistant isolates. The mutational analysis revealed that the most frequent mutations associated with rifampicin and isoniazid resistance in M. tuberculosis are the substitutions at codons 531 (82.7%) and 315 (98.4%) in the rpoB and katG genes, respectively. In addition, we have found mutations with lower frequency at codon 526 (8.4%), 533 (1.5%), and 516 (1.1%) in the rpoB gene. In 6.2% of the isolates, no mutations were found in the rpoB gene. The findings of this study provide useful data for a better understanding of the mutation spectrum of isoniazid and rifampicin resistance among strains isolated from patients in Kazakhstan. Our results are also useful for the development of diagnostic tests of MDR M. tuberculosis.