Background: Neisseria gonorrhoeae infection is a major cause of sexually transmitted disease (STD) and remains a major concern in general and community health. Over the past three decades, there have been reports of N. gonorrhoeae strains resistant to penicillins, tetracyclines, and quinolones from different countries. The purpose of this study was to investigate N. gonorrhoeae drug resistance in view of the widespread use of quinolone antibiotics to low cost and availability, and to search for mutation-induced resistance in order to avoid inappropriate drug use and relapse of infection. Method: This study included 300 women who were referred to obstetrics and gynecology clinics for abnormal vaginal discharge between October 2012 to December 2014 at educational hospitals of Shahid Beheshti University of Medical Sciences, Tehran, Iran. Cultures were prepared for N. gonorrhoeae and positive samples were used to develop an antibiogram. Polymerase chain reaction (PCR) and DNA sequencing were used to study genetic resistance to quinolones. Results: Of 300 specimens included in the study, 7 (2.3 %) were positive for N. gonorrhoeae. Resistance was determined using a disc diffusion method, with ciprofloxacin resistance (57.1%). A minimum inhibitory concentration (MIC) of ≥1 μg/ml showed moderate resistance compared to ciprofloxacin, and an MIC ≥0.25 ± 0.125 μg/ml indicated intermediate resistance. In this study MIC of one sample was 1 μg/ml, 5 samples 0.25 μg/ml, and 1 sample 0.125 μg/ ml. Genotyping of gyrA and parC genes identified a point mutation that induced resistance at an MIC ≥1 μg/ml in the amino acid 86 of gyrA gene and serine to leucin. Conclusion: In the present study, we found that N. gonorrhoeae resistance to quinolones is due to mutation in gyrA gene, as reported by other studies worldwide, as well as in Iran.