Multidrug resistance (MDR) is a common trait developed by many organisms to counteract chemicals and/or drugs used against them. The basic MDR mechanism is relying on an overexpressed efflux transport system that actively expulses the toxic agent outside the cell. In fungi, MDR (or PDR) has been extensively studied in Saccharomyces cerevisiae and Candida albicans, but also plant pathogenic fungi, e.g., Botrytis cinerea, Oculimacula yallundae and Mycosphaerella graminicola are concerned by this phenomenon. In agriculture, the occurrence of MDR strains may threaten the efficacy of current fungicide treatments MDR strains were detected in septoria leaf blotch (M. graminicola) field populations since 2008. These strains are cross-resistant to fungicides with different modes of action. The identification of the molecular mechanism explaining the MDR phenotype in two isolated strains (MDR6 and MDR7) was the main goal of this study. Measuring the intracellular accumulation of a radiolabeled fungicide demonstrated increased fungicide efflux in both MDR strains in comparison to sensitive strains. RNA-sequencing led to the identification of several overexpressed transporter genes, out of which MgMFS1 encoding an MFS (major facilitator family) transporter had particularly abundant mRNA in both MDR strains. Crosses between both MDR strains showed that mdr6 and mdr7 loci are closely linked. We applied bulk-progeny sequencing to progeny of the crosses MDR6 x sensitive and MDR7 x sensitive in order to map the genomic regions co-segregating with the MDR phenotypes. SNP frequency analysis in sensitive and resistant bulks showed a clear co-segregation between phenotypes and the left arm of chromosome 7. This region harbors several genes including the MgMFS1 gene mentioned above with a 514 bp promoter insertion in both MDR strains. Gene disruption of MgMFS1 in the MDR6 strain abolished resistance to several fungicides and reduced the resistance to others, demonstrating the importance of MgMFS1 in the MDR phenotype in M. graminicola. Studies are underway to demonstrate if the promoter insertion by its own is sufficient or if an additional mutation is necessary to drive MgMFS1 overexpression and hence MDR in M. graminicola. Financial support: Arvalis Institut du Vegetal, BASF Agro SAS, Bayer SAS, DuPont de Nemours SAS, Syngenta Crop Protection AG. (MDR) is a common trait developed by many organisms to counteract chemicals and/or drugs used against them. The basic MDR mechanism is relying on an overexpressed efflux transport system that actively expulses the toxic agent outside the cell. In fungi, MDR (or PDR) has been extensively studied in Saccharomyces cerevisiae and Candida albicans, but also plant pathogenic fungi, e.g., Botrytis cinerea, Oculimacula yallundae and Mycosphaerella graminicola are concerned by this phenomenon. In agriculture, the occurrence of MDR strains may threaten the efficacy of current fungicide treatments MDR strains were detected in septoria leaf blotch (M. graminicola) field populations since 2008. These strains are cross-resistant to fungicides with different modes of action. The identification of the molecular mechanism explaining the MDR phenotype in two isolated strains (MDR6 and MDR7) was the main goal of this study. Measuring the intracellular accumulation of a radiolabeled fungicide demonstrated increased fungicide efflux in both MDR strains in comparison to sensitive strains. RNA-sequencing led to the identification of several overexpressed transporter genes, out of which MgMFS1 encoding an MFS (major facilitator family) transporter had particularly abundant mRNA in both MDR strains. Crosses between both MDR strains showed that mdr6 and mdr7 loci are closely linked. We applied bulk-progeny sequencing to progeny of the crosses MDR6 x sensitive and MDR7 x sensitive in order to map the genomic regions co-segregating with the MDR phenotypes. SNP frequency analysis in sensitive and resistant bulks showed a clear co-segregation between phenotypes and the left arm of chromosome 7. This region harbors several genes including the MgMFS1 gene mentioned above with a 514 bp promoter insertion in both MDR strains. Gene disruption of MgMFS1 in the MDR6 strain abolished resistance to several fungicides and reduced the resistance to others, demonstrating the importance of MgMFS1 in the MDR phenotype in M. graminicola. Studies are underway to demonstrate if the promoter insertion by its own is sufficient or if an additional mutation is necessary to drive MgMFS1 overexpression and hence MDR in M. graminicola. Financial support: Arvalis Institut du Vegetal, BASF Agro SAS, Bayer SAS, DuPont de Nemours SAS, Syngenta Crop Protection AG.