Background: Canada conducts surveillance of penicillin, tetracycline, erythromycin, spectinomycin, ciprofloxacin, cefixime, and ceftriaxone susceptibilities in Neisseria gonorrhoeae isolates to support development of national treatment guidelines for sexually transmitted infections. Methods: N. gonorrhoeae isolates were collected by Canadian provincial public health laboratories and included isolates from males and females ranging in age from 1 to 86 years. Minimum inhibitory concentrations (MICs) were determined by agar dilution at the National Microbiology Laboratory, Public Health Agency of Canada, and MIC interpretations were based on the criteria of the Clinical Laboratory Standards Institute. RESULTS:: From 2000 to 2009, 40,875 isolates of N. gonorrhoeae were tested by provincial laboratories and 10,993 of these were characterized by the Public Health Agency of Canada. There was an increasing incidence of N. gonorrhoeae isolates that were chromosomally resistant to penicillin, tetracycline, and erythromycin while the plasmid-mediated resistance strains (penicillinase-producing N. gonorrhoeae, tetracycline-resistant N. gonorrhoeae, and PP/tetracycline-resistant N. gonorrhoeae strain all had a declining trend. The percentage of isolates resistant to ciprofloxacin significantly increased from 1.3% in 2000 to 25.5% in 2009. Only 0.17% of isolates tested were azithromycin resistant. Between 2000 and 2009, the modal MICs for ceftriaxone increased from 0.016 μg/mL to 0.063 μg/mL. Conclusions: Ciprofloxacin resistance in N. gonorrhoeae within Canada has increased to a level where quinolones are no longer the preferred drugs for the treatment of gonococcal infections and the modal MICs for the third-generation cephalosporins have increased over time. Close monitoring of antibiotic susceptibilities are required to inform treatment options.