Endophytic fungi play an important role in plant growth. The composition and structure of endophytes vary in different plant tissues, which are specific habitats for endophyte colonization. To analyze the diversity and structural composition of endophytic fungi from toothed clubmoss (Huperzia serrata) that was artificially cultivated for 3 years, we investigated endophytic fungi from the roots, stems and leaves using comparative sequence analysis of the ITS2 region of the fungal rRNA genes sequenced with high-throughput sequencing technology. Seven fungal phyla were identified, and fungal diversity and structure varied across different tissues, with the most distinctive community features found in the roots. A total of 555 operational taxonomic units (OTUs) were detected, and 198 were common to all samples, and 43, 16, 16 OTUs were unique to the root, stem, leaf samples, respectively. Taxonomic classification showed that Ascomycota and Basidiomycota were dominant phyla, and Cladosporium, Oidiodendron, Phyllosticta, Sebacina and Ilyonectria were dominant genera. The relative abundance heat map at the genus level suggested that H. serrata had characteristic endophytic fungal microbiomes. Line discriminant analysis effect size analysis and principal coordinate analysis demonstrated that fungal communities were tissue-type and tissue-site specific. Overall, our study provides new insights into the complex composition of endophytic fungi in H. serrata.