The threat of different abiotic unfavorable factors to mushroom production necessitates the search for superior strains currently in use in the industry. The most appropriate technology for this aim appears to be mutagenesis. The aim of this study is to examine the effects of physical mutagen (irradiation) on the evolution of oyster mushroom strains with a particular emphasis on heat tolerance. TUFC30034, TUFC100146, TUFC33106, TUFC34869, TUFC30044, TUFC12458, and TUFC30088, Pleurotus ostreatus fungal cultures, were studied at (25, 30, and 35°C) temperatures based on colony morphology and mycelial growth rate. The findings revealed that different strains are sensitive to heat stress in different ways. In comparison to the other strains, TUFC 30034 and TUFC 30044 demonstrated higher heat tolerance. TUFC 34869 had a texture that was somewhat tolerating, regularly shaped, and compact. The candidate strain (TUFC34869) was also grown on a sawdust/rice bran medium to look at yield indices such as mycelium running, pinhead initiation, 1st harvest, and total yield. Due to its performance at various temperatures as well as vegetative features, TUFC 34869 was chosen for further mutation testing. After being incubated at (25, 30, and 35°C), three monosporic mutants were isolated from UV-irradiated cultures, although they demonstrated retarded mycelial growth as compared to their parental strain at 35°C. A more comprehensive study strategy is required, as well as testing the effects of UV on a larger number of Pleurotus strains.