Daptomycin-resistant (DAP-R) Staphylococcus aureus strains are well documented, but have not been reported in China. To elucidate the evolution adaptability and fitness cost of DAP-R S. aureus, three DAP susceptible strains, Pre3 (MRSA, ST239-t037), Pre5 (MRSA, ST239-t037), and Pre14b (MSSA, ST188-t189), were isolated from patients with bloodstream infections, and serially passaged in Mueller–Hinton broth with a gradient of DAP concentration to select for resistance. Highly DAP-R mutants were obtained after screening for 34 passages. The DAP minimum inhibitory concentrations increased from 0.5 μg/ml in the parent strains to 16 μg/ml in the mutants, which remained tolerant to 4 μg/ml of DAP for more than 160 generations. The growth of the three mutant strains was slower than that of the parent strains, with relative fitness cost of 34.8%, 19.2%, and 15.0%, respectively. The in vitro serum tolerance of the mutants was decreased, and the lethality and pathogenicity in mice were weakened (P < 0.01). Transmission electron microscopy found that the cell walls of the mutants were significantly thicker (from 38.6% to 75.4%) than those of the parent cells. Mutation L826F of mprF was found in Post14b, G299V, and L473I of mprF and Y225N of walK were found in Post3, while T345A of mprF, S52N of graS, and F473I of walK were found in Post5. Thus, stable DAP-R mutants could be obtained from a middle-short term of in vitro DAP selection, and according to their fitness cost, some prevention and control work may be done to cope with DAP-R S. aureus that may appear in China in the future.