Abstract: Daptomycin is an antibiotic used to treat infections caused by multidrug-resistant gram-positive pathogens. With increasing resistance to other antibiotics, an increase in daptomycin consumption is expected, which poses a potential threat of emergence of antibiotic resistance. These facts explain the need to study possible mechanisms for resistance emergence. In vitro selection of resistance to daptomycin in methicillin-resistant Staphylococcus aureus SA0420 of the ST239 genetic line was carried out. After 40 passages, the derivative strain was characterized by an increase in daptomycin minimum inhibitory concentration (MIC) from 1 to 64 µg/mL and a decrease in the doubling time and growth rate, while the induced autolytic activity did not change. During the selection and assessment of population diversity by the whole genome sequencing, both homo-mutations (nucleotide substitutions in 96–100% of reads after the alignment with the control genome at the particular position) and hetero-mutations (5‒95% of alternative reads) were revealed. After the fifth passage, the mutation in the S295L codon of the mprF gene was identified. The 40th passage revealed an additional mutation in the R50C codon; this mutation was not previously described. A new variant of the A214T mutation was detected in the Cls2 protein. The hetero-mutation in the Q493K codon of the walK regulatory gene associated with the cell wall was identified during the fifth passage and was not detected during further passaging. Hetero-mutations were also found in the ktrA and ntpA genes encoding the potassium uptake protein and phosphate transport protein, respectively. By the 40th passage of the selection, stop codons in the hypothetical gene yhfP (SACOL1927) were identified. Therefore, daptomycin resistance arises through the development of hetero-populations. After the 5th, 20th, and 40th passages, homo- and hetero-mutations in the genes with the proved role in the emergence of daptomycin resistance (mprF, cls2, and walK), as well as in the potential candidates (ktrA, ntpA, and yhfP), were revealed.