ABSTRACT A rapid emergence of azole resistance has been observed in Aspergillus fumigatus in The Netherlands over the past decade. The dominant resistance mechanism appears to be of environmental origin and involves the TR ₃₄ /L98H mutations in cyp51A . This resistance mechanism is now also increasingly being found in other countries. Therefore, genetic markers were used to gain more insights into the origin and spread of this genotype. Studies of 142 European isolates revealed that those with the TR ₃₄ /L98H resistance mechanism showed less genetic variation than azole-susceptible isolates or those with a different genetic basis of resistance and were assigned to only four CSP (putative cell surface protein) types. Sexual crossing experiments demonstrated that TR ₃₄ /L98H isolates could outcross with azole-susceptible isolates of different genetic backgrounds, suggesting that TR ₃₄ /L98H isolates can undergo the sexual cycle in nature. Overall, our findings suggest a common ancestor of the TR ₃₄ /L98H mechanism and subsequent migration of isolates harboring TR ₃₄ /L98H across Europe.