Aspergillus fumigatus is a ubiquitous opportunistic fungal pathogen that can cause aspergillosis in humans. Over the last decade there have been increasing global reports of treatment failure due to triazole resistance. An emerging hypothesis states that agricultural triazole fungicide use causes clinical triazole resistance. Here we test this hypothesis in Hamilton, Ontario, Canada, by examining a total of 195 agricultural, urban, and clinical isolates using 9 highly polymorphic microsatellite markers. For each isolate, the in vitro susceptibilities to itraconazole and voriconazole, 2 triazole drugs commonly used in the management of patients, were also determined. Our analyses suggested frequent gene flow among the agricultural, urban environmental, and clinical populations of A. fumigatus and found evidence for widespread sexual recombination within and among the different populations. Interestingly, all 195 isolates analyzed in this study were susceptible to both triazoles tested. However, compared with the urban population, agricultural and clinical populations showed significantly reduced susceptibility to itraconazole and voriconazole, consistent with ecological niche-specific selective pressures on A. fumigatus populations in Hamilton. Frequent gene flow and genetic recombination among these populations suggest greater attention should be paid to monitor A. fumigatus populations in Hamilton and other similar jurisdictions.