During 2020–2021, cultivated red-fleshed dragon fruit (Hylocereus polyrhizus) in Phatthalung province, southern Thailand, was infected with canker disease in all stages of growth. Small, circular, sunken, orange cankers first developed on the cladodes of H. polyrhizus and later expanded and became gray scabs with masses of pycnidia. The fungi were isolated using the tissue transplanting method and identified based on the growth of the fungal colony, and the dimensions of the conidia were measured. Their species level was confirmed with the molecular study of multiple DNA sequences, and their pathogenicity was tested using the agar plug method. Morphological characterization and molecular identification of the internal transcribed spacer (ITS), translation elongation factor 1-α (tef1-α) and β-tubulin (tub) sequences revealed the fungal pathogen to be a new species. It was named Neoscytalidium hylocereum sp. nov. The biota of the new species, N. hylocereum, was deposited in Mycobank, and the species was assigned accession number 838004. The pathogenicity test was performed to fulfil Koch’s postulates. N. hylocereum showed sunken orange cankers with a mass of conidia similar to those observed in the field. To our knowledge, this is the first report of H. polyrhizus as a host of the new species N. hylocereum causing stem cankers in Thailand.