d Azithromycin (AZM) is routinely recommended as a component of dual therapy for gonorrhea in combination with third-generation cephalosporins (3GC). In this study, we examined the prevalence of AZM-resistant (AZM r ) Neisseria gonorrhoeae from July 2010 to February 2013, assessed the rate of concurrent cephalosporin resistance under the current treatment recommendations, and analyzed the clonal distribution of AZM r isolates in Ontario, Canada. Nineteen AZM r clinical isolates (one per patient; MIC, >2 g/ml) were included in the study. Susceptibility profiles of these isolates to 11 antibiotics, molecular typing, characterization of macrolide resistance mechanisms, and penicillin-binding protein 2 (PBP2) patterns were determined for all the isolates. Two groups were defined based on AZM r level; group A isolates displayed high-level resistance (MIC, >2,048 g/ml) due to mutations (A2143G) in the four copies of the 23S rRNA rrl gene, and group B isolates had moderate resistance to AZM (MICs, 2 to 8 g/ml, C2599T mutation in the rrl gene), with a subgroup belonging to sequence type 3158 (ST3158) (n ؍ 8), which also showed reduced susceptibility to 3GC (MICs, 0.12 to 0.25 g/ml, PBP2 pattern XXXIV). This AZM r phenotype was not observed in previous provincial surveillance in 2008 (the ST3158 clone was found, with AZM MICs of 0.25 to 0.5 g/ml associated with mtrR mutations). We hypothesized that the AZM mutant prevention concentration (MPC) in the ST3158 subpopulation we found in 2008 was higher than the MPC in wild-type isolates (AZM MIC, <0.031 g/ml), increasing the chances of additional selection of AZM r mutations. Full AZM resistance is now emerging in this clone together with reduced susceptibility to 3GC, threatening the future efficacy of these antibiotics as therapeutic options for treatment of gonorrhea.