One of the main problems for conservation of germplasm of edible mushrooms is that the traditional method of subculturing facilitates aging and contamination of strains. The conservation at ultra-low temperature in liquid nitrogen (-196 °C) is a widely used method, however, it becomes expensive in collections with large numbers of strains and requires highly specialized personnel. Cryopreservation at -80 °C of mycelium on cereal grains (spawn) has also been shown to be an efficient and low cost method for Agaricus species. It could be used for other edible mushrooms. The aim of this study was to evaluate the viability of spawn of Pleurotus pulmonarius and Lentinula edodes strains frozen at -20 or -80°C. Two strains of each species were studied. The spawn was prepared in sorghum seeds and incubated for 3 weeks at 25 ºC to allow the grains were completely covered by mycelium. Fully-incubated sorghum grains were placed in polycarbonate vials containing 1.5 ml of sterile cryoprotectant solution (10% glycerol v/v) or in vials without solution. The grains remained in contact with the cryoprotectant solution for 1 h at room temperature and then vials of both treatments were transferred directly into freezers at -20 or -80 ºC. After 3, 6 and 12 months the samples were thawed for 10 min in a bath water at 30 °C and the viability of the spawn grains was evaluated. Mycelial growth rate was measured on potato dextrose agar (PDA) at 25 °C for 7 days. The percentage of sample recovery was recorded from the number of spawn grains giving rise to a mycelial colony. Recovered mycelia were further used in small scale cultivation experiments to check their fruiting ability. In the samples frozen at -20 °C the recovery was 24 - 100 % and 4 - 94 % for Pleurotus and Lentinula strains respectively. However, the samples frozen at -80 °C with or without cryoprotectant showed a recovery of 100% from 1 to 5 days after thawing. The recovered samples had a normal mycelial growth in PDA. In all cases the recovered samples produced normal basidiomata when cultivated on substrate prepared with pasteurized barley straw. The proposed method may facilitate handling and reduce maintenance for edible mushroom strains.