Background: Pseudomonas aeruginosa has developed resistance to multiple antibiotics. Unfortunately, ciprofloxacin-resistant P.aeruginosa has emerged with multiple resistance mechanisms. Objectives: This study aims to determine the frequency of gyrA (Topoisomerase II) mutation and its contribution to ciprofloxacin resistance in P. aeruginosa isolates from patients at Zagazig University Hospitals. Methodology: Cultivation of pus specimens, identification of isolates by Gram stain, oxidase test and API20NE. Antibiotic susceptibility testing and ciprofloxacin minimal inhibitory concentration (MIC) were done for all isolates. PCR –RFLP was used to detect mutation in gyrA gene. Results: Among 192 examined specimens, 30 (15.6%) P. aerguinosa were isolated mainly from Surgery and Burn Unit. Fourteen isolates (46.7%) were resistant to ciprofloxacin. Ten (71.4%, P<0.001) isolates had shown mutation in gyr A gene by PCR-RFLP. Conclusion: Mutation in gyrA gene is a major mechanism of ciprofloxacin resistance in P. aeruginosa.