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Cherry leaf spot is one of the most common and devastating diseases of cherries worldwide. The disease causes considerable yield losses in many cherry growing regions. We surveyed cherry leaf spot disease in Beijing City and collected 67 fungal isolates from approximately 60 diseased leaves. Multigene phylogenetic analyses coupled with morphological observations facilitated the identification of species isolated from the diseased tissues. Pathogenicity assays were conducted for six isolates representing all the identified species and Koch's postulates were confirmed on three cultivars of Prunus avium under greenhouse conditions. These results confirmed their pathogenicity on cherry leaves as symptoms were reproduced. Based on these results, a novel taxon Alternaria prunicola sp. nov. is reported as the main pathogen of Cherry leaf spot in Beijing City. In addition, Alternaria alternata, Alternaria pseudoeichhorniae sp. nov., Colletotrichum aenigma, Colletotrichum pseudotheobromicola sp. nov., Epicoccum pseudokeratinophilum sp. nov., Nothophoma pruni sp. nov., Nothophoma quercina and Stagonosporopsis citrulli were also isolated from Cherry leaf spots. Significant variations in the virulence were observed among fungal species on different cherry cultivars.