Septoria Leaf Blotch is an important wheat disease caused by Zymoseptoria tritici, mainly controlled by fungicides in the field. Over the last decades, target-site resistance has increased in Z. tritici populations. In parallel, resistance involving enhanced efflux of multiple classes of fungicides has also raised, named multidrug resistance (MDR). MDR in Z. tritici was shown to be driven by the overexpression of the membrane efflux pump encoding gene MFS1, due to the presence of various inserts in its promoter (Omrane et al. 2015, 2017). These inserts are classified type I, IIa/b, or III and their lengths and insertion sites vary.In order to estimate if MDR in Z. tritici was driven by MFS1 promoter inserts solely, we performed a large scale screen of MDR isolates. 475 potential Z. tritici MDR strains were isolated from different regions of France and Europe in 2020 and 2021, through a screen using a spore germination test on media with medical fungicides, tolnaftate and terbinafine. We determined terbinafine resistance levels for all strains as well as MFS1 promoter alleles. Type I insert was found in 59% of isolates, type II in 20%, type III in 5%. Interestingly, 13% displayed the WT promoter allele. Furthermore, qualitative and quantitative phenotyping results of terbinafine resistance showed discrepancies between resistance level of isolates with the same MFS1 promoter allele. This indicates that new and unknown mutations are involved in the MDR phenotype in some isolates. Some of them have an impact on MFS1 expression, studied by RT-qPCR, while others cause an increase in MDR level, independent from MFS1 expression indicating the involvement of additional and undescribed mechanism(s) leading to MDR in Z. 862 16 863 TH EUROPEAN CONFERENCE ON FUNGAL GENETICStritici. Fungicide efflux studies and expression analysis of membrane transporter genes are underway to confirm the MDR phenotype and to identify the transporter(s) involved in increased resistance