A longitudinal study combining multilocus sequence typing with molecular evolutionary analysis determined the distribution, population structure, and evolution of antibiotic resistance in Neisseria gonorrhoeae isolates in Saskatchewan that were collected between 2005 and 2008. Of 195 gonococcal isolates examined, 29 sequence types (STs) were identified with 3 major circulating strains (ST-1 through ST-3) comprising 52% of all gonococcal isolates studied. The prevalences, persistence, distribution patterns, and clonalities of these isolates strongly suggest that gonorrhea endemicity within this broad geographic region was driven by these 3 circulating strains. ST-1 exhibited a significantly (P ؍ 0.001) higher prevalence throughout the study than did the others, accounting for ϳ25% of the tested isolates each year. The spatial distributions of the gonococcal strains indicated that ST-1 in 2007 entered a linear component of the sexual network, reaching the remote north and resulting in the further spread and maintenance of infection. Ciprofloxacin and azithromycin resistances were observed in distantly related gonococcal lineages, clearly indicating the convergent acquisition of these antibiotic-resistant phenotypes. In addition, all ciprofloxacinand azithromycin-resistant lineages were found at the edges of the minimum spanning tree, far from the major lineages, suggesting that these antibiotic phenotypes were most likely introduced into the province. In contrast, resistance to penicillin was found mostly in the endemic gonococcal lineages, suggesting that penicillin resistance was probably acquired in Saskatchewan as a result of spontaneous mutations in already-established lineages. Tetracycline resistance was present in all STs except one, indicating its ubiquitous nature in the gonococcal population studied. N eisseria gonorrhoeae, a fastidious Gram-negative diplococcus, is an obligate human pathogen that causes the disease gonorrhea. Complications from the spread of the organism along the mucosal surfaces may result in endometritis, salpingitis, and peritonitis in females (3), which can lead to infertility or ectopic pregnancy (27). In Canada, between 1999 and 2008, the overall rate of gonococcal infection increased by 116.5%, from 17.6 to 38 cases per 100,000 (20). Saskatchewan has had the highest infection rate of all the Canadian provinces, with an increase from 45.2 cases per 100,000 in 2000 to 131.3 cases per 100,000 in 2008, resulting in a 290.4% increase (173.4% above the national average) (20). Antimicrobial resistance in N. gonorrhoeae is a major public health problem, as treatment options are becoming increasingly limited. Recently, N. gonorrhoeae isolates with decreased susceptibilities (MIC Ն 0.25) to third-generation cephalosporins (cefixime and ceftriaxone) have been identified in Japan, Europe, the United States, and Canada (4,15,16,19,29,30). These antibiotics are the last remaining options for treating gonorrhea in a single-dose fashion, a preferred mode of treatment for gonococcal infection...