[Objective] Researched the diversity and distribution characteristics of endophytic fungi in Nicotiana tabacum in Dali District. [Methods] These strains were identified into genera based on morphological characteristics combined wtih ITS-rDNA gene sequences. [Results] A total of 1 586 strains of endophytic fungi were isolated from the leaves of Nicotiana tabacum and were identified into 31 genera. Alternaria and Fusarium are dominant fungal endophytes, occupying 40.79% and 18.73%, of total isolated strains respectively. [Conclusion] The quantity, diversity and distribution of the endophytic fungi were varied from different part leaves and different districts of N. tabacum. Larger quantity of endophytic fungi was found in lower part leaves than that of the upper ones. Otherwise, richer diversities of endophytic fungi were found in upper leaves. Along with the development of N. tabacum, the richness and diversity of endophytic fungi in N. tabacum leaves were increased and reached the largest during the squaring stage and maturity stage.