Plasmid-mediated quinolone resistance genes in clinical strains cannot be detected by phenotypic traits but require gene detection. We developed a multiplex real-time polymerase chain reaction (PCR) assay using high-resolution melting master mix with ResoLight dye to detect qnr genes and a simplex real-time PCR assay using SYBR Green I to detect qepA genes. Using qnr-positive and qepA1-positive control strains, the ResoLight method was able to rapidly identify qnrA, qnrB, qnrS, qnrC, and qnrD genes; the SYBR Green I method identified qepA genes. Among 118 extended spectrum beta-lactamase-producing Enterobacteriaceae isolates, the 2 new assays efficiently detected and identified qnr in 9 strains, but no qepA gene. To our knowledge, this is the first study describing the detection of all 5 qnr and qepA genes using real-time PCR. The 2 tests constitute a significant step forward for screening for plasmid quinolone resistance genes in clinical strains. Copyright © 2011 Elsevier Inc. All rights reserved.