Cryopreservation of ectomycorrhizal fungi is often difficult, but needed for patent deposits and for enabling experiments on model strains with constant characteristics. In this study we report a protocol for successful cryopreservation of the ectomycorrhizal mushroom Cantharellus cibarius, together with a brief summary of some unsuccessful attempts. A slow freezing rate (0.3°C min−1) of agar cubes carrying the mycelium, and a gentle addition of the cryoprotectants sorbitol and DMSO were important features. This protocol was successfully repeated by ATCC who used it to deposit a patented strain of C. cibarius, and therefore the protocol should also be tested for other ectomycorrhizal basidiomycetes.