Gentamicin is one of the components of combination therapy for infective endocarditis caused by Staphylococcus aureus, including methicillin-resistant S.aureus (MRSA). The purpose of this study was to analyze the effect of ten 6-hour cycles of exposure to high concentrations (16 µg/ml) of gentamicin in vitro on the phenotype and genotype changes of aminoglycoside-susceptible S.aureus strains belonging to four sequence types (ST): ST5 (ATCC 29213), ST8, ST97, and ST22 (MRSA). After selection, an increase in the MIC of gentamicin to 8–64 µg/ml for all strains except ATCC 29213 was observed. One strain (SA0937) dissociated into three morphotypes, including a small colony variant (SCV). A derivative strain SA0937 variant with normal colony size was characterized by associated resistance to daptomycin due to the P314L mutation in MprF. Except for the SCV morphotype, there was no change in growth rate in response to the formation of resistance. After selection, the emergence of the ATCC 29213 strain tolerance was noted. It manifested by an increase in effective killing up to 14 hours in a 24-hour time-killing test with 16 µg/ml of antibiotic concentration. It was revealed that the ATCC 29213 strain has mutations in peptidyl-tRNA hydrolase (Pth). Deletions in the atpG gene, which is part of the ATP synthase complex, were found in three strains. Deletions and mutations in the menaquinone metabolism genes hepS, menA, and translation elongation factor G (fusA) were identified in the remaining derivative strains. Thus, the use of gentamicin is linked to possible rapid development of resistance and tolerance, which is not associated with the acquisition of aminoglycoside-modifying enzyme genes. Detection of SCV is associated with adverse clinical outcomes. It is important to consider the possibility of developing resistance to daptomycin due to gentamicin selection when using combination therapy. © 2023 Publishing House OKI. All rights reserved.