The prevalence of resistance to fusidic acid in clinical isolates of Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), has increased in the past 2 decades. However, there are limited data regarding the relative importance in this process of the different staphylococcal determinants that mediate resistance to fusidic acid. Furthermore, the roles played by clonal dissemination of fusidic acid-resistant strains versus horizontal transmission of fusidic acid resistance determinants have not been investigated in detail. To gain insight into both issues, we examined fusidic acid resistance in 1,639 MRSA isolates collected in Denmark between 2003 and 2005. Resistance to fusidic acid (MIC, >1 μg/ml) was exhibited by 291 (17.6%) isolates. For the majority of these isolates (∼87%), resistance was attributed to carriage of fusB or fusC, while the remainder harbored mutations in the gene (fusA) encoding the drug target (EF-G). The CC80-MRSA-IV clone carrying fusB accounted for ∼61% of the resistant isolates in this collection, while a single CC5 clone harboring fusC represented ∼12% of the resistant strains. These findings emphasize the importance of clonal dissemination of fusidic acid resistance within European MRSA strains. Nonetheless, the distribution of fusB and fusC across several genetic lineages, and their presence on multiple genetic elements, indicates that horizontal transmission of fusidic acid resistance genes has also played an important role in the increasing prevalence of fusidic acid resistance in MRSA.