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Coix lachrymal-jobi L. var. ma-yuen Stapf of Gramineae are annual or perennial herbs and an important food-medicine homologous plants of high value in nutrition, health protection, and comprehensive utilization. In recent years, the revival of researches on its roles in food and medicinal applications of this underutilized grass for food security and economic empowerment of rural communities has been seen . In this research, Coix kernel endophytic fungi were isolated and identified by fungal colony morphology observation combined with the PCR-amplified fungal internal transcribed spacer (ITS) sequence analyses. All together six isolates to five species of Coix endophytic fungi and two isolates to the genus level were identified from the kernels of six Coix cultivars: Penicillium expansum, Penicillium polonicum, Cladosporium cladosporioides, Alternaria alternata, Aspergillus flavus, and two genera of Aspergillus and Fusarium. Potential benefits and harms analyses showed that Penicillium expansum, Aspergillus oryzae, and Cladosporium cladosporioides can produce a variety of beneficial composite enzymes and have an extensive application in microbial chemistry, food science, and fermentation, whereas Penicillium, Aspergillus flavus, Alternaria alternate, and Fusarium can produce corresponding toxins harmful to plants, animals, and humans. These results not only provided a basis for the targeted prevention of contamination in the tissue culture of Coix kernels by the addition of specific antibiotics, but also enriched the endophytic fungi resource pool of Gramineae crops and suggested new ideas for the improvement, cultivation, post-harvest seeds/kernels storage, and the development of new natural drugs.