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Pseudomonas aeruginosa is a clinically significant opportunistic pathogen which rarely causes disease in healthy immunocompetent individuals. The emergence of multi-drugresistant strains in P. aeruginosa isolates has increased worlwide. Fluoroquinolones act as bactericidal agents by inhibiting DNA gyrase and topoisomerase IV, thus inhibiting DNA transcription and replication. The second-generation quinolones have broader clinical applications in the treatment of complicated urinary tract infections and pyelonephritis, sexually transmitted diseases, selected pneumonias and skin infections. The presence of second-generation quinolone resistance-associated alterations in topoisomerase II: gyrA and topoisomerase IV: par C genes was investigated for 54 P. aeruginosa clinical isolates with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses. Our study revealed that gyrA (Thr-83→Ile) mutation was found in 51.5% of ciprofloxacin resistant samples, while it was absent in ciprofloxacin sensitive samples. Accordingly, parC (Ser-87→Leu) mutation was found in 42.4% of ciprofloxacin resistant samples and parC mutations mostly accompanied gyrA mutation. GyrA (Thr-83→Ile) and parC (Ser-87→Leu) mutations were also present in the same value of 33.3% of ofloxacin resistant samples. Higher level of quinolone resistance rates stemming from target site mutations can guide us to take advantage of these molecular surveillance tests for the efficient management of Pseudomonas infections.