Mupirocin has been used against Gram-positive pathogenic bacteria, and is a specific inhibitor of bacterial isoleucyl-tRNA synthetase. In this work, we have determined the prevalence of mupirocin resistance among staphylococci isolated from a Korean hospital, and have investigated the characteristics of the resistance. In Staphylococcus aureus, the prevalence of high-level mupirocin resistance was 5% (16 of 319), whereas low-level mupirocin resistance was not detected. In coagulase-negative staphylococci (CoNS) the rates of high- and low-level mupirocin resistance were 16.7% (34 of 204) and 10.3% (21 of 204), respectively. The high-level resistant strains contained the ileS-2 gene, which encodes a novel staphylococcal isoleucyl-tRNA synthetase. In contrast, all of the low-level mupirocin-resistant CoNS contained the mutation V588F, which is located near the conserved motif KMSKS, within the chromosomal staphylococcal isoleucyl-tRNA synthetase gene (ileS). In conclusion, this work describes the recent, but rapid, emergence of two different types of mupirocin-resistant staphylococci in Korea, and the sequence and mutant characterization of the isoleucyl-tRNA synthetase of CoNS.