Ciprofloxacin comprise a medically effective and generally utilized class of broad-spectrum antibiotics; as a result of the intensive use of this type of therapy, bacteria have become resistant, lead to minimize the usage of such bactericides in field of management as well as treatment of microbial illness. Pseudomonas aeruginosa one of important pathogens that case wide spectrum of infections, the evolution of ciprofloxacin-resistant Pseudomonas aeruginosa were notify. In this investigation 50 clinical isolates of Pseudomonas aeruginosa were collected, antibiotic susceptibility test for ciprofloxacin by using the Kirby–Bauer technique as well as MIC was performed to all these isolates. Specific primers sequences for detection of Topoisomerase II (gyrA) genes were used by conventional PCR then, PCR product were treated with Sac II enzyme at 37°C for 2 h, then restriction bands were recovered by gel electrophoresis. From Fifty Pseudomonas aeruginosa isolates enrolled in this study, 33(66%) show sensitive to Ciprofloxacin, 17(34%) reported resistant to Ciprofloxacin. RFLP-PCR showed that out of 17 isolates resistant to Ciprofloxacin mutation inDNA gyrase (GyrA) were detected in six (35.2 %) isolated while the reminder 11 (64.7%) has no mutation. The presence of mutations in Topoisomerases II genes mediate Ciprofloxacin defeat as therapy for Pseudomonas aeruginosa. The current venture, were made to demonstrate Topoisomerase II genes (GyrA mutations in Pseudomonas aeruginosa that could be responsible for molecular mechanism of Ciprofloxacin resistance.