A strain of the opportunistic pathogenic yeast was genetically modified for use as a cellular model for assessing by allele replacement the impact of lanosterol C14α-demethylase mutations on azole resistance. was chosen because it is susceptible to azole antifungals, it belongs to the CTG clade of yeast, which includes most of the species pathogenic for humans, and it is haploid and easily amenable to genetic transformation and molecular modeling. In this work, allelic replacement is targeted at the locus by the reconstitution of a functional auxotrophic marker in the 3' intergenic region of Homologous and heterologous alleles are expressed from the resident promoter of , allowing accurate comparison of the phenotypic change in azole susceptibility. As a proof of concept, we successfully expressed in different alleles, either bearing or not bearing mutations retrieved from a clinical context, from two phylogenetically distant yeasts, and constitutes a high-fidelity expression system, giving specific Erg11p-dependent fluconazole MICs very close to those observed with the donor strain. This work led us to characterize the phenotypic effect of two kinds of mutation: mutation conferring decreased fluconazole susceptibility in a species-specific manner and mutation conferring fluconazole resistance in several yeast species. In particular, a missense mutation affecting amino acid K143 of Erg11p in species, and the equivalent position K151 in , plays a critical role in fluconazole resistance.< Réduire