The agricultural sector faces the increasing emergence and development of fungicide resistance in Zymoseptoria tritici, the agent of septoria leaf blotch in wheat. Since the early 2010s, strains with 'Multi-Drug Resistance' (MDR) phenotype have been detected in natural populations. This phenotype has been associated with the overexpression of the membrane efflux pump gene MFS1, resulting in increased efflux of fungicides. In this context, understanding the evolution and spread of resistances in this fungus is crucial to protect the effectiveness of treatments.Recent experimental evolution studies, aiming to investigate the selection of resistance mechanisms under various fungicide application conditions, have led to the selection of resistant strains. Surprisingly, some of them display MDR phenotype, which does not seem to be linked to known MFS1 expression regulation. This suggests that previously undescribed MDR mechanisms may have been selected through these experimental evolutions.To elucidate whether increased efflux is involved in these phenotypes, two distinct efflux tests were carried out on a selection of 53 MDR isolates. The first test assessed the efflux of a fluorescent molecule (Nile Red), while the second test investigated the interaction between terbinafine (fungicide) and transport modulators (inhibitors of ABC and MFS transporters) on fungal growth. The majority of the studied isolates exhibited an increase in Nile Red efflux in comparison to the ancestor sensitive strain and/or clear synergy between at least one transport modulator and terbinafine on growth inhibition. This suggests that their MDR phenotype may be attributed to increased fungicide efflux. Other isolates showed different phenotypes, indicating the involvement of alternative MDR mechanisms, not necessarily through increased efflux.Additionally, whole-genome sequencing data are currently being analysed and may provide further insights into the molecular mechanisms underlying MDR