| Summary: | European hornbeam (<i>Carpinus betulus</i> L.) is widely planted in landscaping. In October 2021 and August 2022, leaf spot was observed on <i>C. betulus</i> in Xuzhou, Jiangsu Province, China. To identify the causal agent of anthracnose disease on <i>C. betulus</i>, 23 isolates were obtained from the symptomatic leaves. Based on ITS sequences and colony morphology, these isolates were divided into four <i>Colletotrichum</i> groups. Koch’s postulates of four <i>Colletotrichum</i> species showed similar symptoms observed in the field. Combining the morphological characteristics and multi-gene phylogenetic analysis of the concatenated sequences of the internal transcribed spacer (ITS) gene, Apn2-Mat1-2 intergenic spacer (ApMat) gene, the calmodulin (CAL) gene, glyceraldehyde3-phosphate dehydrogenase (GAPDH) gene, Glutamine synthetase (GS) gene, and beta-tubulin 2 (TUB2) genes, the four <i>Colletotrichum</i> groups were identified as <i>C. gloeosporioides</i>, <i>C. fructicola</i>, <i>C. aenigma</i>, and <i>C. siamense</i>. This study is the first report of four <i>Colletotrichum</i> species causing leaf spot on European hornbeam in China, and it provides clear pathogen information for the further evaluation of the disease control strategies.
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