[Objective] Diversity of fungal endophytes associated with Juglans regia was studied. [Methods] Fungal endophytes from different seasons, habitats and parts of Juglans regia were isolated by tissue expand method. Strains were classified by morphology and similarity of internal transcribed spacer (ITS) sequence by ClustalX 2.0. Composition, diversity and preference of fungal endophytes were analyzed by the colonization frequency (CF), isolation rate (IR), isolation frequency (IF), Shannon-Wiener biodiversity index (H′). [Results] In total 1 450 fungal endophytes were isolated from Juglans regia and classified into 47 genera. Among them, Alternaria (20.00%), Fusarium (8.34%), Saccharomyces (8.14%) were the dominant genera. The diversity of fungal endophytes and Evenness index (E) were maximum in 2-yesr-old twig, Shanyang, spring with Shannon-Wiener index 1.15, 1.42, 1.92 and evenness 0.36, 0.42, 0.54, respectively. [Conclusion] There was some difference in diversity of fungal endophytes isolated from different parts, habitats, season of Juglans regia. Meanwhile, the similarity coefficient and evenness show that the fungal endophytes isolated from Juglans regia were relatively stable.