The study aimed at determining the presence of qnr genes in ESBL-positive strains of Klebsiella pneumoniae isolated from clinical material obtained from patients hospitalized in several intensive care units in the University Hospital Olomouc.The study comprised 100 ESBL-producing Klebsiella pneumoniae isolates from clinical samples obtained from patients hospitalized between 1 January 2008 and 31 January 2011. blaTEM, blaSHV, blaCTX-M and qnr genes were detected by polymerase chain reaction (PCR). To compare the similarity or identity of the isolates, pulsed-field gel electrophoresis (PFGE) of DNA fragments was used.The blaSHV gene was detected in 99 from 100 isolates of Klebsiella pneumoniae, which is in accordance with the published data that suppose the chromosomal position of the blaSHV-1 gene in this species. The blaCTX-M gene was detected in 77 isolates. The qnr genes were revealed in 56 isolates and majority (76.8 %) of these qnr-positive bacteria carried also the genes encoding the beta-lactamases CTX-M and TEM. In all cases, the qnrB variant was detected. Plasmids from nine different incompatibility groups were identified, in most cases from the IncFII group (52 isolates). Antibiotic susceptibility tests revealed high frequency of resistance not only to beta-lactams, but also to aminoglycosides, tetracycline and sulfamethoxazole/trimethoprim. PFGE detected 65 different strains and several groups of isolates with the same restriction profile. Although the clinical significance of the qnr genes is not well established, their presence in ESBL-positive Enterobacteriaceae is not negligible and it should be kept in mind.