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. Plant pathogenic fungi are also concerned by this phenomenon. MDR strains were detected in septoria leaf blotch (Zymoseptoria tritici) field populations since 2008. These strains are cross-resistant to fungicides with different modes of action due to active fungicide efflux. In a previous study, we identified the MFS1 gene overexpressed in all tested MDR field strains (1). This gene encodes a major facilitator membrane transporter whose inactivation abolished the MDR phenotype in two resistant isolates (MDR6 and MDR7). To identify the mutation(s) responsible for MDR phenotype we applied bulkprogeny sequencing to crosses involving MDR6 and MDR7 strains. This analysis enabled us to identify a 519 bp insert in the MFS1 promoter in both strains. The insert, a reminiscence of a recent retrotransposition event, is responsible for MFS1 overexpression and the MDR phenotype. Genotyping of various field strains revealed that at least one additional mutation is responsible for the MDR phenotype.