| Summary: | <i>Celtis julianae</i> Schneid. is widely planted as a versatile tree species with ecological and economic significance. In September 2022, a leaf blotch disease of <i>C. julianae</i> was observed in Nanjing, Jiangsu, China, with an infection incidence of 63%. The disease led to severe early defoliation, significantly affecting the ornamental and ecological value of the host tree. The accurate identification of pathogens is imperative to conducting further research and advancing disease control. Koch’s postulates confirmed that the fungal isolates (B1–B9) were pathogenic to <i>C. julianae</i>. The morphology of the characteristics of the pathogen matched those of <i>Alternaria</i> spp. The internal transcribed spacer region (ITS), large subunit (<i>LSU</i>) and small subunit (<i>SSU</i>) regions of rRNA, glyceraldehyde-3-phosphate dehydrogenase (<i>GAPDH</i>), <i>Alternaria</i> major allergen gene (<i>Alt a 1</i>), RNA polymerase second largest subunit (<i>RPB2</i>), and portions of translation elongation factor 1-alpha (<i>TEF1-α</i>) genes were sequenced. Based on multi-locus phylogenetic analyses and morphology, the pathogenic fungi were identified as <i>Alternaria arborescens</i> and <i>A. italica</i>. The findings provided useful information for disease management and enhanced the understanding of <i>Alternaria</i> species diversity in China. This is the first report of <i>A. arborescens</i> and <i>A. italica</i> causing leaf blotch of <i>C. julianae</i> in China and worldwide.
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