Daptomycin is a lipopeptide antibiotic used clinically for the treatment of methicillin-resistant Staphylococcus aureus (MRSA). Emergence of daptomycin non-susceptible S. aureus during therapy is often associated with multiple genetic changes, however, the relative contribution of these changes to resistance and other phenotypic changes often remain unclear. The present study was undertaken to investigate this issue using a genetically characterized series of four isogenic clinical MRSA strains derived from a patient with bacteremia, before and during daptomycin treatment. The first strain obtained after daptomycin therapy carried a single nucleotide polymorphism (SNP) in rpoB (RpoB A477D) that decreased susceptibility not only to daptomycin but also to vancomycin, beta-lactams, and rifampicin. Furthermore, the rpoB mutant exhibited pleiotropic phenotypes including increased cell wall thickness, reduced expression of virulence traits, induced expression of the stress-associated transcriptional regulator, Spx, and slow growth. A subsequent acquired loss-of-function mutation in clpX partly alleviated the growth defect conferred by the rpoB mutation without changing antibiotic susceptibility. The final isolate had acquired three additional mutations including a SNP in mprF (MprF S295L) known to confer daptomycin non-susceptibility, and accordingly this isolate was the only daptomycin non-susceptible strain of this series. Interestingly, in this isolate, the cell wall had regained the same thickness as the parental strain, while transcription of the vraSR (cell wall stress regulator) was increased. In conclusion, this study illustrates how serial genetic changes selected in vivo contribute to daptomycin non-susceptibility, growth fitness and virulence traits.