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 has been extensively studied in Saccharomyces cerevisiae and Candida albicans, but also plant pathogenic fungi are concerned by this phenomenon. In this study, we confirmed a drug efflux based resistance mechanism in two isolates of the septoria leaf blotch agent Mycosphaerella graminicola. Using next generation sequencing approaches, we could demonstrate that the MgMFS1 gene encoding a membrane transporter of the major-facilitatorsuperfamily was strongly overexpressed in both isolates. The mutation(s) responsible for the MDR phenotype was/were mapped by a bulk-progeny-sequencing approach to a 38.5 kb genomic fragment on chromosome VII, covering also the MgMFS1 gene. Inactivation of the MgMFS1 gene the MDR6 strain abolished the MDR phenotype showing the involvement of this transporter in drug resistance. However fine mapping is necessary to identify the mdr mutation(s) leading to MgMFS1 overexpression.