Truffles (Tuber spp.) are the most precious ectomycorrhizal edible mushrooms whose biodiversity is seriously endangered.To develop a protocol for cryopreservation of Tuber spp. mycelia using T. borchii as a model species, verifying whether conservation in liquid nitrogen may affect viability, growth rate, hyphal morphology and infectivity.Cryopreservation was performed using sorbitol, sucrose and DMSO as cryoprotectants. The morphological parameters analyzed were: hyphal diameter, septal distance and hyphal growth unit. Cryopreserved mycelium infectivity was assessed by inoculating Quercus robur seedlings.In T. borchii cryopreserved mycelium, the lag-phase lasted for 6-42 days but no differences in growth curve evolution, growth rate and hyphal morphology were observed except for hyphal growth unit. No differences in mycorrhizal colonization were observed between the seedlings inoculated with non-cryopreserved and cryopreserved mycelium.The established protocol is suitable for long-term conservation of Tuber mycelium and opens up the possibility of creating a Tuber spp. germplasm bank to preserve truffle diversity.