【Objective】 To confirm the pathogen species that caused brown rot disease on Hylocereus undatus. 【Method】 Diseased tissue isolation, indoor pathogenicity test, morphological observation and rDNA sequence analysis were used to confirm the pathogen species. 【Result and conclusion】 The colonies of the fungus were dark grey and fluffy on potato dextrose agar (PDA) medium. Two kinds of arthrospore were observed on PDA medium. One was columnar conidium with light-colored, thin-walled and (5.00-10.69) μm×(2.61-4.52) μm in size, and the other was chained conidium with dark brown, thick-walled, circular or oval, base truncate and (5.15-12.39) μm×(3.87-6.07) μm in size. Using PCR with primers ITS1 and ITS4 for internal transcribed spacer (ITS) sequences of fungal 18S-28S rDNA，the sequence was cloned to be 579 bp in length. The BLAST results showed that the sequence had the highest sequence identity with that of Neoscytalidium dimidiatum strains isolated from pitahaya (99%-100%). These results indicated that night-blooming cereus brown rot in Guangdong was caused by N. dimidiatum.