© 2021 Elsevier Inc.Thermophilic fungi have several biotechnological and industrial applications such as thermostable enzyme production, biodegradation, and tobacco processing, etc. Thermophilic fungi cannot survive at temperatures below 20 °C. Owing to their inability to grow at low temperatures, they are not stable, so stocking is very difficult. Although a large number of different storage methods are available and described, no method can be universally applied to all fungi. Thermophilic fungi present “heat-loving” characteristics, and therefore a new challenge for its preservation and there is no universal protocol for the preservation of thermophilic fungi. The aim of this study was to evaluate the viability, contamination and stability of thermophilic fungi stored under different preservation methods. In this work, 25 thermophilic fungal isolates of species Thermomyces thermophilus, Rhizomucor pusillus, Trichocladium griseum, Melanocarpus albomyces, Malbranchea cinnamomea, Thermothelomyces thermophilus, Thermothelomyces hinnuleus, Thermothielavioides terrestris, Mycothermus thermophilus, Humicola insolens maintained constant sub-culturing at room temperature, +4 °C and +20 °C, lyophilization at +4 °C, freezing at −20 °C, freezing block at −20 °C and a new technique liquid preservation at room temperature for the periods ranging 5 years. We evaluated the effect of preservation methods by sub-culturing onto either sabouraud dextrose agar (SDA) or yeast extract soluble starch agar (YpSs) on growth, production and viability of spores and macro- and micromorphology. In this study, preservation methods for thermophilic fungi were investigated extensively for the first time and it is clearly shown that freezing block at −20 °C method and lyophilization were better methods for long-term preservation up to 5 years.