AbstractSuccinate dehydrogenase inhibitor (SDHI) fungicides are widely used for the control of a broad range of fungal diseases. This has been the most rapidly expanding fungicide group in terms of new molecules discovered and introduced for agricultural use over the past fifteen years. A particular pattern of differential sensitivity (resistance) to a subclass of chemically-related SDHIs (SHA-SDHIs) was observed in naïveZymoseptoria triticipopulations. Class specific SHA-SDHI resistance was confirmed at the enzyme level but did not correlate with the genotypes of the succinate dehydrogenase (SDH) encoding genes. Mapping and characterization of the genetic factor responsible for standing SHA-SDHI resistance in natural field isolates identified a gene (alt-SDHC) encoding a paralog of the C subunit of succinate dehydrogenase. This paralog was not present within our sensitive reference isolates and found at variable frequencies withinZ. triticipopulations. Using reverse genetics, we showed that alt-SDHC associates with the three other SDH subunits leading to a fully functional enzyme and that a unique Qp-site residue within the alt-SDHC protein confers SHA-SDHI resistance. Enzymatic assays, computational modelling and docking simulations for the two types of SQR enzymes (alt-SDHC, SDHC) enabled us to describe protein-inhibitor interactions at an atomistic level and to propose rational explanations for differential potency and resistance across SHA-SDHIs. EuropeanZ. triticipopulations displayed a presence (20-30%) / absence polymorphism ofalt-SDHC, as well as differences inalt-SDHCexpression levels and splicing efficiency. These polymorphisms have a strong impact on SHA-SDHI resistance phenotypes. Characterization of thealt-SDHCpromoter in EuropeanZ. triticipopulations suggest that transposon insertions are associated with the strongest resistance phenotypes. These results establish that a dispensable paralogous gene determines SHA-SDHIs fungicide resistance in natural populations ofZ. tritici. This study paves the way to an increased awareness of the role of fungicidal target paralogs in resistance to fungicides and demonstrates the paramount importance of population genomics in fungicide discovery.Author SummaryZymoseptoria triticiis the causal agent of Septoria tritici leaf blotch (STB) of wheat, the most devastating disease for cereal production in Europe. Multiple succinate dehydrogenase inhibitor (SDHI) fungicides have been developed and introduced for the control of STB. We report the discovery and detailed characterization of a paralog of the C subunit of the SDH enzyme conferring standing resistance towards a particular chemical subclass of the SDHIs. The resistance gene is characterized by its presence/absence, expression and splicing polymorphisms which in turn affect resistance levels. The identified mechanism influenced the chemical optimization phase which led to the discovery of pydiflumetofen, exemplifying the importance of population genomics for discovery and rational design of the most adapted solutions.