Abstract: 【Objective】The study created new strains of Pleurotus giganteus through monokaryon hybridization method, and combined with germplasm resource evaluation, in orde to provide high temperature resistant and excellent germplasm resources for mushroom industry in tropical regions. 【Method】Based on strains source statistics, high temperature tolerance testing and agronomic traits evaluation, PG46 and PG79 with excellent and complementary agronomic traits and obvious differences in high temperature tolerance were selected as hybrid parents. Monokaryon strains were obtained through protoplast monokaryonization and single spore separation method, and the mating types of monokaryons were identified. Monokaryon strains from different parents with different mating types were hybridized through confrontational culture, then the new strains were screened and obtained by means of microscopically detecting clamp connection structure and antagonistic lines. At the same time, high-temperature tolerance tests, agronomic traits evaluation,and nutritional component determination of fruit body were conducted on hybrid strains, to screen out new P. giganteus germplasms with high temperature tolerance, high yield, high quality for cultivation in tropical regions. 【Result】With the method of protoplast monokaryonization and single spore separation, 14 monokaryon strains from PG46 were obtained,including 7 A₁B₁ mating types and 7 A₂B₂ mating types; 8 monokaryon strains from PG79 were obtained, including 2 A₁B₁ mating types and 6 A₂B₂ mating types. A total of 28 hybrid combinations were obtained through monokaryons pairing, and 15 hybrid strains were identified with clamp connections through microscopy, of which 9 strains had better mycelial growth. Furthermore,the germplasm resource evaluation was conducted on 9 hybrid strains and 2 parent strains, and a new hybrid strain PGH6 was ultimately selected with high temperature tolerance（40 ℃ stress for 18 h could still recover growth）, short growth period（53 d, 12 and 20 d shorter than their parents）, good commercial traits（round and regular cap, short stipe, and storage resistance）, and high nutritional component（crude protein content 13.13 g/100 g, crude fiber content 17.93 g/100 g, amino acid content 7.57 g/100 g）. 【Conclusion】A new hybrid strain with high yield,high quality, and high temperature tolerance is obtained through monokaryon hybridization breeding, providing new germplasm for P. giganteus industry in tropical regions of China.