Scedosporium spp. cause infections (scedosporiosis) in both immunocompetent and immunocompromised individuals and may persistently colonize the respiratory tract in patients with cystic fibrosis (CF). They are less susceptible against azoles than are other molds, such as Aspergillus spp., suggesting the presence of resistance mechanisms. It can be hypothesized that the decreased susceptibility of Scedosporium spp. to azoles is also CYP51 dependent. Analysis of the Scedosporium apiospermum and Scedosporiumaurantiacum genomes revealed one CYP51 gene encoding the 14-α-lanosterol demethylase. This gene from 159 clinical or environmental Scedosporium isolates and three Lomentospora prolificans isolates has been sequenced and analyzed. The Scedosporium CYP51 protein clustered with the group of known CYP51B orthologues and showed species-specific polymorphisms. A tandem repeat in the 5' upstream region of Scedosporium CYP51 like that in Aspergillus fumigatus could not be detected. Species-specific amino acid alterations in CYP51 of Scedosporium boydii, Scedosporiumellipsoideum, Scedosporium dehoogii, and Scedosporiumminutisporum isolates were located at positions that have not been described as having an impact on azole susceptibility. In contrast, two of the three Sapiospermum-specific amino acid changes (Y136F and G464S) corresponded to respective mutations in A. fumigatus CYP51A at amino acid positions 121 and 448 (Y121F and G448S, respectively) that had been linked to azole resistance.