Background: Globally, Neisseria gonorrhoeae antimicrobial resistance has been increasing, and in particular, reports of isolates with reduced susceptibility to third-generation cephalosporins have surfaced. We examined the phenotypic and genetic characteristics of 155 N. gonorrhoeae isolates with decreased susceptibilities to third-generation cephalosporins isolated in Canada between 2001 and mid-2010. Methods: Minimum inhibitory concentrations (MICs) were determined by agar dilution on N. gonorrhoeae isolates, and those displaying elevated MICs to cefixime (MIC = 0.25 μg/mL and 0.5 μg/mL) and ceftriaxone (MIC = 0.125 μg/mL and 0.25 μg/mL) were examined using N. gonorrhoeae multiantigen sequence typing (NG-MAST) and sequencing of resistance determinants associated with decreased cephalosporin susceptibilities (penA, mtrR, ponA, porB1b (penB alteration). Results: Between 2001 and 2010, there has been a shift in the modal MICs from 0.016 to 0.125 μg/mL for cefixime and from 0.016 to 0.063 μg/mL for ceftriaxone. Thirty-seven different sequence types (STs) were identified among the isolates using N. gonorrhoeae multiantigen sequence typing; ST3158, ST225, and ST1407 were most prevalent at 25.9%, 19.4%, and 14.8%, respectively. The penA mosaic was present in 60% of the isolates, with the most common penA mosaic types XXXII and X identified at 51.0% and 7.7%, respectively, whereas the nonmosaic penA type XII was identified in 36.8% of the isolates. Conclusions: In Canada, N. gonorrhoeae isolates with decreased susceptibilities to third-generation cephalosporins have increased over the years. The alterations in penA, mtrR, and porB1b (penB alteration) are important determinants identified in these isolates. The most common STs identified among these Canadian isolates have also been reported worldwide.