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Six collections of ascomycetes were obtained from samples collected from dead branches and leaves of Juglans regia in Guizhou and Yunnan provinces, China. By incorporating multigene phylogenetic analysis (ITS, LSU, rpb2, SSU, tef1-α, tub2) supplemented by morphological data, we establish two novel species, namely Helminthosporium guizhouense and Nigrospora yunnanensis. In morphology, H. guizhouense can be distinguished from H. caespitosum by its narrower conidia (13–16 µm vs. 27.3–35.5 µm), and N. yunnanensis is characterized by black, globose conidia (16.2 × 14.4 µm). The phylogenetic results further substantiated them as novel taxa. The present study contributes to our comprehension of the range of fungi found in Juglans regia, thereby expanding our knowledge of the diversity of fungi within this host.