Beauveria bassiana is an entomopathogenic fungus that is widely used in Thailand to control pest insects. However, the increasing temperature has influenced the insect control efficiency of the fungus. Therefore, determination of thermotolerant isolates of B. bassiana that can grow and remain pathogenic at higher temperatures than its current optimum temperature may be a better way to control pest insects in a high temperature environment. Three isolates of B. bassiana obtained from the Rice Department, Thailand were selected for mutagenesis using ethyl methanesulfonate (EMS) with subsequent screening at high temperatures (33 and 35°C). In addition, the recovery of fungal growth after exposure to a high temperature for a period of time (5–15 days) and then transferring to 25°C was evaluated. No isolates were found that grew at 35°C but one mutant isolate (BCNT002MT) produced larger diameter colonies and more spores than the corresponding wild type (WT) at 33°C. Growth and spore production of the BCNT002MT isolate were greater than its WT when incubated at 25°C for 14 days following exposure to 33°C for 7 days. In addition, the spore germination level (%) of BCNT002MT was significantly higher than its WT during culture at 25°C after prior exposure to 33°C for 5, 10 and 15 days. The pathogenicity against the brown planthopper, Nilaparvata lugens (Stål), of this mutant isolate was also prominent.