Abstract: To clarify the pathogen causing E. breviscapus leaf spot in Yunnan Province along with its biological characteristics. Leaves exhibiting typical symptoms of the leaf spot disease were collected for the purposes of pathogen isolation, identification and pathogenicity assessment. The morphological characteristics of the pathogens were observed, and their rDNA-ITS, LSU, and Act gene sequences were analyzed. Additionally, the biological characteristics of the pathogen were determined. The results indicated that the symptoms of the leaf spot were nearly circular and brown, exhibiting a verticillate pattern. The colonies of the purified isolates appeared grayish-green, while the hyphae ranged from white to grayish-brown.. The pycnidium is brown, spherical or flattened, size 61.54～91.68 × 106.31～173.84 μm; The Conidia are single cells that are oval or elliptical in shape, transparent without septum, with oil droplets, 4.87～7.39 × 1.55～4.26 μm in size. Combining the results of morphological characteristics and phylogenetic analysis, the pathogen causing E. breviscapus leaf spot in Yunnan Province was identified as D. segeticola. Biological characteristics indicated that soluble starch is the optimal carbon source for strain YB1,while beef extract serves as the best nitrogen source.The optimum growth temperature is 28 ℃, and the optimum pH range was 6～8, light conditions were found to be most favorable for mycelia growth, PDA was identified as the best culture medium among 6 media tested. The mycelia did not grow after being subjected to a continuous water bath at 72 ℃ for 10 min. This study provides a scientific basis for the diagnosis and control of E. breviscapus leaf spot disease.