We investigated the prevalence of mutations in the gyrA, gyrB, grlA, and grlB genes in 21 clinical isolates of methicillin-resistant Staphylococcus aureus recovered from ocular infections. These isolates were classified into 16 groups on the basis of pulsed-field gel electrophoresis (PFGE) patterns. Among these nonidentical strains, 9 different combinations of mutations were observed. We observed an unusually high frequency of mutations: 10 (62.5%) of the isolates had four or five mutations, 5 (31.3%) of the isolates had double mutations, and 1 isolate had a single mutation. Different mutations were detected in strains of the same PFGE type, and this is consistent with the independent acquisition of mutations. The minimun inhibitory concentrations values ranged from 0.5 to 512 microg/mL for levofloxacin and 0.125 to 128 microg/mL for gatifloxacin. Only 1 isolate was found to be susceptible to both agents. These results suggest that application of high concentration fluoroquinolone ophthalmic solutions selects for isolates presenting such mutations that may result in a high level of resistance to fluoroquinolones.