Resistance to quinolone has increased significantly and one of the most reasons is plasmid-mediated quinolone resistance (PMQR). The aim of this study is to detect the prevalence of PMQR in multidrug-resistant (MDR) Gram negative bacilli and to characterize these resistance genes. A total of 420 Gram negative bacilli clinical isolates were collected from patients attending Misr children hospital. Isolates were identified by biochemical reactions, while antimicrobial susceptibility testingwas done by Kirby-Bauer disk diffusion method. Minimum inhibitory concentrations (MIC) of ciprofloxacin were detected by E-test, whereas combined test method was used to detect extended-spectrum β-lactamase (ESBL) production. QnrA, qnrB, and qnrS genes were determined by multiplex polymerase chain reaction (PCR). MDRGram negative bacilli represented 68% (268/420); most of them were recovered from blood culture specimens (21%).Among these MDR isolates21%(60/268) were ciprofloxacin resistant; with MICs >32µg/ml in 95% of the isolates.ESBL production was detected in 11.7% of the studied isolates. The qnr genes were detected in 60%. QnrS and qnrB were the detected genes in 77.8% and 16.7% of the isolates respectively. Both qnrB and qnrS genes were determined simultaneously in 5.5%.QnrB gene was found alone in only one isolate (14.3%) that was ESBL-producer. The most MDR isolates were recovered from blood culture; this confirms the occurrence of these superbugs and their ability to cause life threatening infections. The prevalence of quinolone resistant Gram negative bacilli clinical isolates is high. The mostly prevalent PMQR gene is qnrS followed by qnrB.