Mol Plant Pathol. 2020;00:1-17. | 1 wileyonlinelibrary.com/journal/mpp 1 | INTRODUC TI ON Sclerotinia sclerotiorum is a plant-parasitic fungus that causes white mould disease. It is known for its aggressive necrotrophic lifestyle, which means that the fungus actively kills the plant host cells and thrives by feeding on the dead plant material, and for exhibiting a broad host range. S. sclerotiorum can infect more than 600 host plants including economically important species such as tomato Abstract Sclerotinia sclerotiorum is a notorious generalist plant pathogen that threatens more than 600 host plants, including wild and cultivated species. The molecular bases underlying the broad compatibility of S. sclerotiorum with its hosts is not fully elucidated. In contrast to higher plants and animals, alternative splicing (AS) is not well studied in plant-pathogenic fungi. AS is a common regulated cellular process that increases cell protein and RNA diversity. In this study, we annotated spliceosome genes in the genome of S. sclerotiorum and characterized their expression in vitro and during the colonization of six host species. Several spliceosome genes were differentially expressed in planta, suggesting that AS was altered during infection. Using stringent parameters, we identified 1,487 S. sclerotiorum genes differentially expressed in planta and exhibiting alternative transcripts. The most common AS events during the colonization of all plants were retained introns and the alternative 3′ receiver site. We identified S. sclerotiorum genes expressed in planta for which (a) the relative accumulation of alternative transcripts varies according to the host being colonized and (b) alternative transcripts harbour distinct protein domains. This notably included 42 genes encoding predicted secreted proteins showing high-confidence AS events. This study indicates that AS events are taking place in the plant pathogenic fungus S. sclerotiorum during the colonization of host plants and could generate functional diversity in the repertoire of proteins secreted by S. sclerotiorum during infection. K E Y W O R D S alternative splicing, computational analysis, host adaptation, isoforms, RNA sequencing (RNA-seq), Sclerotinia sclerotiorum