Pleurotus tuoliensis (Bailinggu) is a precious mushroom species cultivated industrially in eastern Asia. The long cropping cycle, high deformity rate, and low yield are the major bottlenecks for its widespread cultivation. There is thus a need for improved P. tuoliensis cultivars. In the present study, interspecific crossing of monokaryons between P. tuoliensis and P. eryngii was carried out. Six out of nine pairings were observed to result in clamp connections. Dikaryotized regions within a mating Petri dish varied considerably with the compatible pairings. A total of nine hybrids were isolated within the junction zone and on the periphery at either sides of the paired colonies from three selected crosses. The internal transcribed spacer (ITS) restriction fragment length polymorphism (ITS-RFLP) profiles, together with ITS sequencing revealed significant genetic differences between the nine selected hybrids with parental strains. The polymerase chain reaction (PCR) amplification revealed that the inheritance of mitochondrial DNA was uniparental between these two species. Of nine crossbred strains, six were selected for fruiting test. Compared with their parental P. tuoliensis strains, all the six tested hybrids fruited faster, and three hybrids showed improved biological efficiency compared to their parents, either P. tuoliensis or P. eryngii. Two hybrids (Ht-26 and He-26) formed a white-colored fruit body, with improved biological efficiency (62.5 %–64.2 %) compared to parents P. tuoliensis (41.8 %) and P. eryngii (52.1 %), and thus hold great potential as new cultivars for the commercial production of P. tuoliensis.