We aimed to generate linezolid-resistant Mycobacterium tuberculosis strains from 10 different, fully susceptible M. tuberculosis parental strains. Ten linezolid-resistant colonies could be isolated from six different parental strains, with the frequency of in vitro appearance ranging from 2×10⁻⁸ to 5×10⁻⁹. MIC values of resistant strains varied from 4 to 32 g/ml (parent strains had 1 g/ml). Genotypic characterization showed mutations in the 23S rRNA gene in five strains: four with G-to-T at position 2061 (MIC 32 g/ml) and one with G-to-T at 2576 (MIC 16 g/ml); five strains with MIC 4-8 g/ml had no mutations. Growth rate investigation revealed that resistant strains with 23S rRNA mutations had elevated time to detection (TTD, 10.10±3.49 days) compared to parental strains (5.93±1.84 days) and resistant strains without mutations (5.81±1.81 days). Isolated mutants can be divided into two classes corresponding to M. smegmatis clones: one with wild-type growth, lower MIC, no 23S rRNA mutation (non-ribosomal mechanism); the other with 23S rRNA alterations, high MIC, decreased growth. The G2061T mutation in M. tuberculosis is reported here for the first time.