The monitoring of antimicrobial susceptibilities in Neisseria gonorrhoeae isolates and characterization of N. gonorrhoeae multiantigen sequence types (NG-MAST, ST) provide important surveillance data as resistance rates continue to rise. A total of 2970 N. gonorrhoeae isolates were collected by Canadian provincial public health laboratories in 2010, and 1233 were submitted to the National Microbiology Laboratory for testing. The NG-MAST and minimum inhibitory concentration (MIC) by agar dilution were determined for each isolate. Of the 2970 isolates, 25.1% were resistant to penicillin, 34.6% resistant to tetracycline, 31.5% resistant to erythromycin, 35.9% resistant to ciprofloxacin, and 1.2% resistant to azithromycin. Decreased susceptibility to cefixime (MIC ≥ 0.25 mg/L) and ceftriaxone (MIC ≥ 0.125 mg/L) was identified in 3.2% and 7.3% of the isolates, respectively. The most common STs found in Canada were ST1407 (13.3%), ST3150 (11.3%), and ST3158 (9.0%), with 249 different STs identified among the isolates. Within the ST1407 group, 19.5% and 43.3% isolates have decreased susceptibility to cefixime and ceftriaxone, respectively. ST1407, the most prevalent NG-MAST in Canada in 2010, has been associated with high-level ceftriaxone MICs and with cefixime treatment failure cases worldwide. Identification and monitoring of STs and corresponding antimicrobial resistance profiles may be useful in surveillance programs and be used to inform public health actions.