Chlorhexidine is an antiseptic agent used for hand hygiene worldwide. To evaluate the susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) to chlorhexidine, this study determined MICs of chlorhexidine and another 12 antimicrobial agents, carriage of the Panton-Valentine leukocidin, qacA/B, and smr genes, genetic relatedness by multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec element type for 206 MRSA clinical isolates from the Taiwan Surveillance of Antimicrobial Resistance program III and IV (years 2002 and 2004) from 26 hospitals. Using MLST, we respectively identified 102 (49.5%), 68 (33.0%), 13 (6.3%), 5 (2.4%), 5 (2.4%), and 13 (6.3%) isolates as ST239, ST59, ST5, ST241, ST573, and other types. The MIC(50) and MIC(90) of chlorhexidine for all 206 isolates were 2 and 8 microg/mL, respectively. Seventy-three (35.4%) isolates carried qacA/B gene, but none carried smr. For the 72 (35.0%) MRSA isolates with chlorhexidine MIC > or =4 microg/mL, 53 were ST239 (49 of them carried qacA gene), 12 were ST5 (all carried qacB gene), 5 were ST241 (4 carried qacA gene), 1 was ST338 (and carried qacA gene), and 1 was ST573 (and carried qacA gene). Compared with other sequence-type MRSA isolates, ST239 MRSA isolates were the most resistant to both chlorhexidine and other antimicrobial agents. Methicillin-resistant S. aureus strains with disinfectant resistance qacA/B genes are common in Taiwan. High frequency of qacA/B genes among specific sequence types (ST239, ST5, and ST241) resulted in low susceptibility to chlorhexidine. Periodic surveillance of antiseptic susceptibility among MRSA isolates is important for the control of nosocomial hospital-acquired infections.