Seed storage is critical for preserving genetic resources, but optimal long-term storage conditions for Miscanthus seeds have not been established. In this five-year study, we evaluated storage protocols by comparing seed germination after four and five years, along with field establishment performance. The results demonstrated that genotype, storage conditions, and the storage duration all significantly influenced germination percentage and vigor index of Miscanthus seeds. Low temperature storage yielded the highest germination percentage (59.44%) and vigor index (132.06) in the 4th year, while low temperature with desiccant gave the highest germination percentage (42.41%) in the 5th year. The field performance after direct sowing was also significantly influenced by genotype and storage conditions, with the highest seedling survival (7.80%) observed under low temperature with desiccant. The seeds stored under low temperature exhibited minor structural damage, with the intact cell membranes, the small intercellular gaps, and the orderly cell arrangement. Through comprehensive evaluation, storage at −18 °C with desiccant was determined to be optimal. Based on these results, we strongly recommend storing Miscanthus seeds at −18 °C with desiccant. This protocol offers a reliable and effective solution for farmers, seed producers, and storage facilities to ensure long-term seed viability.