Low thermotolerance in entomopathogenic fungi is the main impediment to their industrialization. This research, for the first time, describes the generation of a thermotolerant colony by pairing and subculturing two Beauveria bassiana isolates without sexual reproduction. A mixture of B. bassiana ERL1578 and ERL1576 was inoculated on 1/4SDAY. The paired culture was subcultured three times, followed by a heat treatment as a selection pressure. Two morphologically different colonies (BbHet1 and BbHet2) were isolated from the pairing. BbHet2 had the most rapid mycelial growth and produced sponge-like mycelial masses (the others were flat), and its conidia were darker than the non-paired colonies under a microscope. BbHet2 conidia had 60.7% germination after exposure to 45°C for 60 min (the others had <15%) without significant loss of virulence against western flower thrips, but a slight decrease in conidial yield was found. Sequencing of Bloc locus and 2-D electrophoresis were done to further clarify the phenomenon. The new phenotypes formed suggested that a genetic variation happened as a result of heterokaryosis and/or recombination, more than environmental adaptation, when mixing different conidia. This methodology seems to be very useful for enhancing thermotolerance in fungi.