Background: quinolone resistance is traditionally mediated by chromosomal mutations mutation of DNA gyrase and/or topoisomerase IV or by the mutation of genes regulating the expression of efflux pumps, until PMQR was described in a clinical isolate of Klebsiella pneumoniae in 1998.PMQR genes generally confer low-level resistance, with their MICs falling below Clinical and Laboratory Standards Institute (CLSI) breakpoints for intermediate resistance; therefore, their contribution to quinolone resistance can be masked in strains also harboring QRDR mutations in gyrA and parC. However, their clinical significance stems from the fact that they greatly facilitate the selection of more highly quinolone-resistant strains. Although the PMQR mechanism only confers low-level resistance to FQs, its association with the occurrence of mutations in QRDR can lead to clinically relevant resistance levels. These PMQR determinants are increasingly being identified worldwide in clinical isolates of Enterobacteriaceae and Pseudomonas spp. Aim of the work: this study aimed to identify different mechanisms of fluoroquinolones resistance and determine fluoroquinolones resistance pattern among the studied isolates. Material and methods: this study was carried on 100 non duplicate clinically relevant Enterobacteriaceae and Pseudomonas spp. recovered from clinical specimens referred to Central Microbiology Laboratory, Ain Shams University Hospital for routine culture and sensitivity, aiming to 1) Determine the occurrence of plasmid-mediated fluoroquinolones resistance (PMQR) determinants by multiplex PCR and chromosomal mutations by PCR-RFLP among Enterobacteriaceae and Pseudomonas spp. in clinical specimens. 2) Identify different mechanisms of Fluoroquinolones resistance. 3) Determine Fluoroquinolones resistance pattern among the studied isolates. Results: in this study we found that 77% of FQs resistant isolates were positive to one or more plasmids, oqxAB was highest recovered PMQR among Klebsiella. 78% were positive for gyrA mutations, gyrA gene mutations were higher in Pseudomonas, Asp-87mutation was 56/78(72%) higher than Ser-83 mutation 38/78 (49%) isolates.