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Human activities promote the rapid adaptation of certain organisms, notably crop pests and pathogens. Active ingredients (AIs), particularly antibiotics and pesticides, have been widely deployed over the 20th and 21st centuries as powerful control tools, favoring the emergence of resistance in target populations. As the renewal of the therapeutic arsenal slows and resistance becomes widespread, it is increasingly urgent to adopt a “One Health” approach to preserve the efficacy of our pharmacopeia, a cornerstone of public health and food security. In this context, we studied the evolution of fungicide resistance in the model fungus Zymoseptoria tritici, the causal agent of septoria leaf blotch in wheat. In particular, we focused on multidrug resistance (MDR), a broad-spectrum generalist resistance phenotype. Associated with moderate resistance factors, its impact on treatment efficacy in agriculture is often underestimated. However, due to its generalist nature, MDR is likely to compromise the effectiveness of resistance management strategies and future AIs. This thesis aims to explore the mechanisms and genomic determinants of MDR in Z. tritici, as a basis for a better understanding of this phenotype. Detected in the field in Europe since 2009, MDR in Z. tritici has been primarily associated with overexpression of the efflux transporter gene Mfs1, driven by transposable element (TE) insertions in its promoter (PMFS1), leading to increased, non-specific efflux of AIs. Recent field monitoring data, however, indicate a quantitative increase in resistance factors, suggesting the contribution of additional mechanisms. To identify these mechanisms, we investigated MDR mutants obtained through experimental evolution under different fungicide selection pressures. Efflux assays revealed contrasting mechanisms depending on the mutant, involving increased efflux or not. Among candidate genes identified by whole-genome resequencing, we identified a gain-of-function C740Y mutation in an AtrR-like transcription factor, which we experimentally validated as contributing to MDR. Furthermore, cases of aneuploidy associated with specific resistance profiles were identified, suggesting that genomic plasticity contributes to adaptive dynamics leading to MDR selection in vitro. We also explored MDR mechanisms in natural strains by analyzing 374 European isolates (2020–2021). Sequencing of PMFS1 revealed TE insertion polymorphism more diverse than previously described, partially associated with the MDR phenotype. Quantification of terbinafine resistance (a phenotypic marker of MDR) in these isolates and their progeny from sexual crosses highlighted the quantitative nature of this trait. Its polygenic basis was confirmed through a genome-wide association study (GWAS), revealing candidate genes with diverse putative functions, consistent with the complex determinism of MDR observed in vitro. This work demonstrates that MDR in Z. tritici can emerge through multiple evolutionary trajectories, leading to the selection of diverse mechanisms, including increased efflux dependent or independent of MFS1 overexpression. It highlights the high genomic plasticity of this fungus, which likely facilitates its rapid adaptation to fungicides, as increasingly reported in other pathogenic fungi. Because limiting the selection of these mechanisms represents a major challenge for sustainable disease control, our study underscores the need to better integrate MDR prevention into resistance management strategies in agriculture.