Abstract: To investigate the biodiversity,distribution,and the microbial community structure of the endophytic fungi in Nicotiana tabacum,the endophytic fungi in different cultispecies were systematically isolated and identified.The preliminary screening of the cytotoxic activities was also performed.As a result,a total of 62 strains of the endophytic fungi were isolated and purified from the healthy tissues (roots,stems,and leaves) of tobacco using the tissue isolation method.The isolated strains were identified to belong to 4 orders,7 families,and 9 genera based on their morphological and molecular characteristics.More species and strains were isolated from the leaves,followed by the stems,and those in the roots were the least.The cytotoxicity of the crude extracts was determined by CCK-8 assay.The results indicated that most of the strains were found to have some inhibitory activities on Caki-1 and HepG2 cell lines.The IC₅₀s of the strain TE-6L to Caki-1 at 24 h and 48 h were 13.28 and 0.36 μg/mL,respectively.Combined with morphological characteristics and molecular analysis,this strain was identified as Fusarium sambucinum.The results from this study indicated that the tobacco possess abundant endophytic fungi,which provides a new path for searching for novel antitumor resources.