Through a survey of rose diseases in the South Tropical Garden in Kunming, China, it was found that black spot was the most common and serious disease of rose cultivated in the open air there, with an incidence of more than 90%. In this study, fungus isolation was performed on leaf samples of five black spot susceptible varieties of rose from the South Tropical Garden by tissue isolation. 18 strains of fungus were initially obtained, and seven of them were finally identified to cause black spot symptoms on healthy leaves of rose after verification by Koch's rule. By observing the morphology of colonies and spores, and constructing a phylogenetic tree by combining molecular biology and multiple genes, two pathogenic fungus were identified, namely,AlternariaalternataandGnomoniopsisrosae.G.rosaewas the first pathogenic fungi of rose black spot isolated and identified in this study. The results of this study can provide a reference base for further research and control of the black spot disease of rose in Kunming.