In order to study the resistance of Neisseria (N.) gonorrhoeae to the fluoroquinolone and detect mutation patterns of quinolone resistance-determining regions (QRDRs) of clinical isolates in Shanghai, China, a total of 80 clinical isolates of N. gonorrhoeae were consecutively collected from Shanghai. The MIC of fluoroquinolone for the isolates was examined by using the agar dilution method and the mutation profiles of the QRDRs of gyrA and parC were analyzed by sequencing and restriction fragment length polymorphism (RFLP). Chi-square test was used for comparison of the mutation patterns. The results showed that: (1) High percentages of the 8 isolates were resistant to ciprofloxacin (95.0%), ofloxacin (95.0%) and lomefloxacin (97.5%), only one strain was susceptible to the ciprofloxacin. (2) Sensitive strains had a substitute of Asp95-->Ala in the gyrA, and all isolates that were resistant or intermediated to the ciprofloxacin, had a double mutation in the gyrA (Ser91, Ala 92 and Asp95). Some strains also had a mutation in the parC. (3) The MICs of these isolates were significantly associated with the mutation patterns in the gyrA and parC. A double mutation of gyrA combined with parC87 mutation was a predominant pattern in Shanghai and could mediate high level resistance to ciprofloxacin. It suggests that mutations in the QRDRs of gyrA and parC may be responsible for the fluoroquinolone resistance. And fluoroquinolone could not be used as the first line antibiotics for gonorrhea treatment any more in Shanghai, China.