Abstract: In order to identify the pathogen of an unknown fungal tobacco leaf spot disease in Fuquan City, Guizhou Province. Illumina Miseq high-throughput sequencing technology was used to analyze the differences of fungal community structure and diversity between healthy and diseased tobacco leaves. The results showed that after tobacco leaves were infected by this disease, the fungal community structure changed, and the fungal community richness and diversity decreased. Sobs index, Ace index, Chao1 index and Shannon index of the fungal community in diseased tobacco leaves were lower than that of healthy tobacco leaves, while Simpson index was higher than that of healthy tobacco leaves. Notability analysis showed that fungal community of healthy tobacco group was significantly higher than that of diseased tobacco group in class (p=0.027), order (p=0.035), family (p=0.026), genus (p=0.021), species (p=0.029) and OTU (p=0.038). The results of Student's t test showded that the relative abundance of Boeremia, Rhodotorula and Unclassified_c_Dothideomycetes were significantly different in healthy and diseased tobacco leaves. Among them, Boeremia was the most dominant genus in the diseased tobacco leaves, and Rhodotorula was the most dominant genus in the healthy tobacco leaves. It is speculated that the unknown fungal leaf spot of tobacco may be caused by Boeremia.