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OBJECTIVES: To determine the frequency of cyp51A variants among a contemporary collection of triazole-resistant Aspergillus fumigatus clinical isolates from the USA and compare their specific contribution to reduced triazole susceptibility.METHODS: The genomes of 87 isolates with decreased susceptibility to at least one mould-active triazole antifungal collected between 2007 and 2020 were sequenced. The cyp51A variants were identified and genetically introduced into a cyp51A-null strain. Antifungal susceptibilities for the mould-active triazole antifungals were determined following CLSI guidelines, and variant positions were mapped after protein homology modelling.RESULTS: Most clinical isolates (60%) carried one of 15 variants that reduced susceptibilities to short-chain (G448S, Y121F, TR46/Y121F/T289A and TR46/Y121F/T289A/N512), long-chain (P216L, F219S and substitutions in the G54 and M220 residues) or multiple (TR34/L98H and TR34/L98H/S297T/F495I) triazoles. While TR34/L98H and TR46/Y121F/T289A were among the most common variants affecting susceptibility, occurring in 13 and 9 isolates, respectively, variants exclusively affecting the coding region and influencing susceptibility were most common, occurring in 31 isolates. A total of 14 isolates (16%) exhibited reduced susceptibility that could not be explained by variants in any known resistance determinants (cyp51A, hmg1 or hapE).CONCLUSIONS: Our findings demonstrate the range of mutations affecting triazole resistance among US isolates and provide a much-needed side-by-side comparison of the impact of these variants on the most currently used mould-active triazole antifungals. Moreover, they underscore the extent to which triazole resistance remains unexplained by known genetic determinants and the need for further discovery.