BACKGROUND: Over the last decade, extremely-drug-resistant (XDR) strains of Klebsiella pneumoniae have emerged worldwide, mainly as a result of patient-to-patient spread. The predominant clone, sequence type 258 (ST258), is associated with high morbidity and mortality, and is a worldwide threat to public health. It was hypothesized that reduced susceptibility to chlorhexidine, the most widely used hospital disinfectant, may contribute to the endemic nature of this strain. AIM: To characterize and compare the susceptibility of the epidemic K. pneumoniae clone ST258 and non-epidemic K. pneumoniae clones to chlorhexidine. METHODS: The minimum inhibitory concentration (MIC) of chlorhexidine was determined in 126 XDR K. pneumoniae clinical isolates using agar dilution. Expression of three different efflux pumps -cepA, acrA and kdeA - was investigated in the absence and presence of chlorhexidine using quantitative real-time polymerase chain reaction. Heteroresistance to chlorhexidine was identified using population analysis. FINDINGS: The MIC of chlorhexidine was higher for K. pneumoniae ST258 (N = 70) than other K. pneumoniae sequence types (N = 56); 99% of ST258 isolates had MICs >32 μg/mL, compared with 52% of other K. pneumoniae sequence types (P < 0.0001). Reduced susceptibility to chlorhexidine appeared to be independent of the expression of cepA, acrA and kdeA efflux pumps. Chlorhexidine-resistant subpopulations were observed independent of the bacterial sequence type or the MIC. CONCLUSIONS: Reduced susceptibility to chlorhexidine may contribute to the success of XDR K. pneumoniae as a nosocomial pathogen, and may provide a selective advantage to the international epidemic strain K. pneumoniae ST258. The heterogeneous nature of chlorhexidine-resistant subpopulations suggests that this phenomenon might not be rendered genetically.