The objective of this study was to develop and validate an expanded multiplex PCR assay for the simultaneous detection of eight plasmid-mediated quinolone-resistance determinants in Enterobacteriaceae. Primers were designed to amplify conserved fragments of qnrABCDS, qepA, oqxAB and aac(6′)-Ib-cr genes and were optimized in uniplex and multiplex PCR assays with control template DNA. The assay was used to determine the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in 174 ciprofloxacin-resistant and 43 ciprofloxacin-susceptible extraintestinal pathogenic Escherichia coli isolates. Each resistance gene could be detected alone and in combination. PMQR determinants were detected in 65 ciprofloxacin-resistant isolates (37 %) and one ciprofloxacin-susceptible isolate (2 %). Prevalences of the identified determinants were: aac(6′)-Ib-cr, 34.5 %; qnrS, 1.1 %; qepA, 1.1 %; and oqxAB, 0.6 %. In conclusion, we developed an eight-target multiplex PCR for the accurate detection of PMQR genes and confirmed that PMQR prevalence remains low among human Escherichia coli clinical isolates in the UK.