Abstract: Seed endophytes play an important role in the growth and development of seeds. In order to understand the structure and diversity of endophytic fungal community in tobacco seeds, the rDNA of ITS region was as the target sequence, the endophytic fungal community structure and diversity of tobacco seeds of varieties K326, Yunyan85 and Yunyan87 were determined and analyzed by the Illumina MiSeq sequencing technology. A total of 342 264 sequences were obtained from tobacco seeds of three varieties, 110 OTUs were obtained by clustering, belonging to 5 phyla, 13 classes, 24 orders, 34 families and 56 genera. The endophytic fungal community structures of K326 and Yunyan 85 were similar, and the diversity was higher than Yunyan 87. Gibberella, Monographella, Alternaria, Fusarium, Rhodotorula, Colletotrichum, Cryptococcus and Aspergillus had high abundance in tobacco seeds. The FUNGuild function prediction analysis showed that the endophytic fungi in tobacco seeds included the following functional groups:plant pathogens, animal pathogens, endophytes, wood saprophytes, lichen parasites, soil saprophytes and wood saprophytes. There were abundant endophytic fungi colonized in tobacco seeds, and the potential beneficial functional fungi were Fusarium, Colletotrichum, Cryptococcus, Gibberella and Aspergillus.