Pleurotus species harbor rich wild germplasm, but commercial cultivation relies on few strains, narrowing genetic diversity. This study domesticated wild Pleurotus djamor (HP), P. pulmonarius (FX), P. ostreatus (CP, CPH) and a commercial P. eryngii (XB), evaluated growth and substrate utilization, performed monokaryotic crossbreeding, and analyzed hybrid transcriptomes. FX showed the fastest mycelial growth and shortest substrate colonization time, while XB was slowest; carbon and nitrogen source preferences were strain-specific, with organic nitrogen generally superior. FX, HP, and XB were selected as parents. From 83 monokaryons and 372 crosses, HP×FX yielded the highest hybridization success; selected hybrids were confirmed by molecular methods. HP×XB hybrids showed severe fruiting abnormalities, whereas HP×FX hybrids produced normal, morphologically distinct fruiting bodies. Transcriptome analysis placed hybrid expression between parents but biased toward FX; enriched functions in hybrids included oxidoreductase and secondary metabolism, while pathway enrichment (endocytosis, cell cycle, proteasome, nucleocytoplasmic transport) mirrored parental differences. These results demonstrate the value of wild Pleurotus germplasm and targeted crossbreeding for creating genetically and phenotypically novel cultivars.