Abstract Agaricus subrufescens is a basidiomycete which is studied because of its medicinal and gastronomic importance; however, less attention has been paid to its preservation. This study aimed to evaluate the effect of sucrose addition to substrate and cryotube on the viability of Agaricus subrufescens cryopreserved at -20 °C and at -75 °C for one and two years. Zero, 10% or 20% sucrose was added to potatodextroseagar or wheat grain. The mycelia were cryopreserved in the absence of cryoprotectant or with sucrosesolutions at 15%, 30% or 45%. After one or two years at -75 °C or at -20 °C, mycelia were thawed and evaluated about viability, initial time of growth, colony diameter and genomic stability. Cryopreservation at -20 °C is not effective to keep mycelial viability of this fungus. Cryopreservation at -75 °C is effective when sucrose is used in substrates and/or cryotubes. Without sucrose, cryopreservation at -75 °C is effective only when wheat grains are used. Physiological characteristic as mycelial colony diameter is negatively affected when potatodextroseagar is used and unaffected when wheat grain is used after two-year cryopreservation at -75 °C. The fungusgenome does not show alteration after two-year cryopreservation at -75 °C.