Background: Acinetobacter baumannii (A. baumannii) is an important organism in hospital acquired infections. A. baumannii has emerged as Quinolones resistant organism.The maximum crucial mechanism of fluoroquinolones resistance is the mutation in genes that encode the subunits of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). Objectives: determination of mutations of gyrA/B and parC/E genes using Restriction Fragment Length Polymorphism PCR (RFLP - PCR) among fluoroquinolones resistant clinical isolates of A. baumannii showed in ICU patients at Al-Azhar University hospitals. Subjects & Methods: The study was conducted on 100 isolates of A.baumannii which subjected to molecular study of mutations of gyrA, gyrB, parC and parE genes beside determination of minimal inhibitory concentration (MIC). Results: The most A. baumannii isolates had MIC >128 µg/ml (61.1%). The resistant isolates showed combind mutations in both gyrA& parC genes most frequently (79.2%) then gyrA (9.7%), parC (8.3%) and gyrB (2.8%). Conclusion: Mutations in gyrA and parC are the most common in fluoroquinolones resistant A. baumannii isolated from Al-Azhar University hospitals