A total of 59 and 74 nonduplicate plasmid-mediated quinolone resistance (PMQR) genes-carrying Klebsiella pneumoniae and Escherichia coli isolates were collected. All strains were assayed for fluoroquinolone susceptibility and the prevalence of quinolone resistance-determining regions (QRDRs) mutation. The association between PMQR determinants and common β-lactamase genes was also analyzed. Genetic relatedness of the isolates was analyzed by pulsed-field gel electrophoresis (PFGE). The PMQR genes-carrying K. pneumoniae and E. coli isolates exhibited high fluoroquinolone resistance rates, indicating that PMQR determinants play an essential role in the development of fluoroquinolone resistance. Remarkably, most qnr-carrying strains had only a single or no QRDR mutation in GyrA or ParC, and most exhibited decreased ciprofloxacin (CIP) susceptibility or low-level CIP resistance. However, 71.4% and 98.4% of qnr-negative K. pneumoniae and E. coli contained double QRDR mutations, and most presented high-level CIP resistance. Additionally, K. pneumoniae presented a lower CIP resistance rate than E. coli (59.3% vs. 91.9%) and low carriage of double QRDR mutations (38.9% vs. 89.9%). Also, most (88.1%) K. pneumoniae examined in this study carried qnr and only 14.9% of E. coli were qnr positive. Thus, the high fluoroquinolone susceptibility of K. pneumoniae isolates is primarily due to fewer QRDR substitutions as a result of high prevalence of qnr alleles in the host. Our findings support the hypothesis that chromosomal resistance mutations could be affected by the presence of Qnr, in other words, Qnr may protect the QRDR domains in the gyrase and topoisomerase IV from mutations under the inhibition of fluoroquinolones. Another remarkable finding was that the PMQR genes-carrying K. pneumoniae exhibited much higher proportions of extended-spectrum β-lactamases (ESBLs)-positive phenotype than E. coli (73.5% vs. 59.5%). This is due to not only the high prevalence of SHV-type ESBL-conferring enzymes in K. pneumoniae but also the interference of DHA-producing K. pneumoniae as a result of the strong association between qnrB and bla(DHA).