A total of 514 consecutive clinical Escherichia coli isolates, irrespective of resistance background, were collected in the period 2002-2008 in Wenzhou, southern China, to investigate the prevalence of plasmid-mediated quinolone resistance (PMQR). The dominant PMQR gene was aac(6')-Ib-cr, followed by qnr, whereas qepA was absent. A total of 253 (49.2%) of these isolates were aac(6')-Ib-positive. Subsequently, 134 of these isolates were sequenced and 42 (31.3%) found to harbor aac(6')-Ib-cr, 18 to harbor new aac(6')-Ib mutants, and 74 to harbor wild-type aac(6')-Ib. The genes qnrA, qnrB, and qnrS were found in 2 (0.4%), 6 (1.2%), and 14 (2.7%) of 514 isolates, respectively, with 2 isolates co-harboring qnrB and qnrS genes. Sequencing allowed us to identify qnrA1, qnrB4, qnrB6, and qnrS1 in 20 qnr-positive isolates, with qnrS1 being the most prevalent allele. The genes qnrC and qnrD were not found in any isolates. Interestingly, 35% of qnr-positive isolates and 16.7% of aac(6')-Ib-cr-positive isolates were susceptible to ciprofloxacin. PMQR genes are therefore present in both quinolone-resistant and -susceptible isolates and can also be transferred by conjugation experiments, thus suggesting a likely future increase in quinolone resistance.