In Malaysia, Lignosus rhinocerus is one of the few important traditional medicinal mushrooms being used by indigenous communities to treat diseases. Currently, this rare mushroom can be found in the deep forest in Peninsular Malaysia, but its number is insufficient to meet the increasing local demand. Therefore, a vitrification technique previously used in the cryopreservation of actinomycetes was adapted in this study to preserve and maintain the commercially potential L. rhinocerus strain in a viable state. In this study, combinations of different sucrose concentrations and exposure time were experimented without serial washing phase after thawing. In addition, electron microscopy and comet assay were applied to study the cryoinjury and genotoxity of vitrified mycelial cells. Mycelial cells incubated for 10 minutes in 1.6 M sucrose of Plant Vitrification Solution 2 (PVS2) yielded largest radial mycelial growth with 100% survival rate. Scanning electron microscopy results indicated the swelling of mycelial cells due to osmotic shock which occurred from thawing procedure, while transmission electron microscopy findings revealed fusion of two nucleus membranes of dikaryotic mycelium. Comet assay suggested insignificant differences (p > 0.05) of comet formation between the normal and vitrified mycelial cells, suggesting cryoprotectants used in vitrification will not cause genotoxity to mycelial cells of L. rhinocerus. In conclusion, the current vitrification technique is suitable to cryopreserve the dikaryotic mycelial cells of L. rhinocerus with 100% regeneration and without trace of genotoxicity.