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Objectives: Azole resistance among Aspergillus fumigatus isolates is a growing concern worldwide. Induction of mutations during azole therapy, environment-acquired mutations caused by azole fungicides and intrinsic resistance of cryptic Fumigati species all contribute to the burden of resistance. However, there is a lack of data in Canada on this emerging threat.Methods: To gain insights into the magnitude and mechanisms of resistance, a 14 year collection of Aspergillus section Fumigati comprising 999 isolates from 807 patients at a Montreal hospital was screened for azole resistance, and resistance mechanisms were investigated with the combined use of genome sequencing, 3D modelling and phenotypic efflux pump assays.Results: Overall azole resistance was low (4/807 patients; 0.5%). A single azole-resistant A. fumigatus sensu stricto strain, isolated from a patient with pulmonary aspergillosis, displayed efflux-pump-mediated resistance. Three patients were colonized or infected with azole-resistant cryptic Fumigati species (one Aspergillus thermomutatus, one Aspergillus lentulus and one Aspergillus turcosus). Evidence is presented that azole resistance is efflux-pump-mediated in the A. turcosus isolate, but not in the A. lentulus and A. thermomutatus isolates.Conclusions: Azole resistance is rare in our geographic area and currently driven by cryptic Fumigati species. Continued surveillance of emergence of resistance is warranted. [ABSTRACT FROM AUTHOR]