To develop a rapid and reliable single-tube-based PCR technique for detecting simultaneously the plasmid-mediated quinolone resistance qnrA, qnrB and qnrS genes. After multiple alignments, primers were designed to detect known qnr variants (six for qnrA-, six for qnrB- and two for qnrS-like genes). They were used for screening a collection of 64 expanded-spectrum beta-lactamase (ESBL)-producing enterobacterial isolates from Kuwait, collected from 2002 to 2004, as ESBL genes have been often associated with qnr genes. Sequencing was performed to identify qnr and associated ESBL genes. In optimized conditions, all positive controls (used separately or mixed) confirmed the specificity of the PCR primers. Out of 64 isolates, only 3 isolates were positive for a qnrB-like gene (4.7%), whereas no qnrA-like and qnrS-like gene was detected. A qnrB2 gene was detected in an Enterobacter cloacae K34 (SHV-12+) isolate, whereas qnrB1-like (termed qnrB7) and qnrB6-like (termed qnrB8) genes were identified from E. cloacae K37 (SHV-12+) and Citrobacter freundii K70 (VEB-1b+) isolates, respectively. We report here a fast and reliable technique for rapid screening of qnr-positive strains to be used for epidemiological surveys. A low prevalence of Qnr determinants among ESBL-producing Enterobacteriaceae was identified in the study with Kuwaiti isolates.