Disease control of septoria leaf blotch (Zymoseptoria tritici) on wheat relies mainly on resistant wheat cultivars and fungicide applications. The fungus, however, displays a high potential to circumvent both methods. Resistance due to target site modifications affects all available uni-site fungicides. A different type of resistance has evolved among Z. tritici populations impacting multiple chemical families. Strains considered as multidrug resistant (MDR) have been isolated since 2008. MDR is a common trait developed by many organisms to counteract chemicals and relies on overexpressed drug efflux transporters that expulse the drug outside the cell. In Z. tritici the major-facilitator gene, MFS1, as principal player of this emerging resistance mechanism is overexpressed in MDR field strains (Omrane et al. 2015). We identified three different types of MFS1 promoter inserts as responsible mutations for MDR in Z. tritici field strains (Omrane et al. 2017). This study aimed to develop a molecular tool allowing to detect and to quantify the frequency of MDR strains in Z. tritici populations. We decided to apply an amplicon-deep sequencing based assay in order to correlate the number of sequencing reads to the frequency of the strains. We chose the Oxford-Nanopore sequencing technology for thelong sequencing read length and its low cost. The principle of the method on complex DNA mixtures is to add unique sequence tags to each molecule in the mixture, prior to PCR amplification to minimize amplification bias. Each population is labeled with a barcode, allowing mixing up to 96 populations per sequencing run. Using this methodology, we successfully detected the four MFS1 promoter alleles in our sequence data of home made DNA mixtures. Further adjustments of the protocol are needed for quantification in complex DNA mixtures and especially in DNA from infected wheat leaves