In this study, the prevalence of the plasmid-mediated quinolone resistance (PMQR) genes among pediatric clinical isolates of Escherichia coli and Klebsiella pneumoniae was investigated. A total of 243 nonduplicate clinical isolates of E. coli and K. pneumoniae collected between January 2010 and December 2012 from Beijing, China, were studied. In total, 55 isolates (22.63%) were positive for PMQR genes, the most frequently detected gene was qnrS (13.2%), followed by aac(6′)-Ib-cr (6.2%) and qnrB (3.7%). The qnrA and qepA genes were not detected. Furthermore, 92.73% (51/55) produced extended-spectrum β-lactamases (ESBLs) and 21.82% (12/55) were resistant to quinolones. DNA sequencing results showed that 14.55% (8/55) of isolates possessed gyrA mutations, while 1.82% (1/55) had parC mutations in the quinolone resistance-determining region (QRDR). Nalidixic acid or ciprofloxacin resistance of the transconjugants increased from 2- to 32-fold. Enterobacterial repetitive intergenic consensus sequence polymerase chain reaction typing indicated that most isolates were not clonally related. Our findings showed that the PMQR detection rate among pediatric clinical isolates of E. coli and K. pneumoniae was high in China. PMQR-positive strains were more common among ESBL-producing and ciprofloxacin-susceptible isolates. Conjugation experiments showed that these isolates could be transferred horizontally. The present study highlights the high prevalence of PMQR in Chinese pediatrics who are not treated with quinolones. © Copyright 2016, Mary Ann Liebert, Inc. 2016.