AbstractFungal pathogens can rapidly evolve virulence towards resistant crops in agricultural ecosystems. Gains in virulence are often mediated by the mutation or deletion of a gene encoding a protein recognized by the plant immune system. However, the loci and the mechanisms of genome evolution enabling rapid virulence evolution are poorly understood. We performed genome-wide association mapping on a global collection of 106 strains of Zymoseptoria tritici, the most damaging pathogen of wheat in Europe, to identify polymorphisms linked to virulence on two wheat varieties. We found 25 distinct genomic loci associated with reproductive success of the pathogen. However, no locus was shared between the host genotypes, suggesting host specialization. The main locus associated with virulence encoded a highly expressed, small secreted protein. Population genomic analyses showed that the gain in virulence was explained by a segregating gene deletion polymorphism. The deletion was likely adaptive by preventing detection of the encoded protein. Comparative genomics of closely related species showed that the locus emerged de novo since speciation. A large cluster of transposable elements in direct proximity to the locus generated extensive rearrangements leading to multiple independent gene losses. Our study demonstrates that rapid turnover in the chromosomal structure of a pathogen can drive host specialization.