【Objective】Our previous studies showed that the Clitopilus hobsonii recovered from ectomycorrhizal root tips in several Quercus species. This fungus was effective in promoting tree growth. The main purpose of this work is to generate the homokaryotic strains of this fungus for obtaining a high-quality genome assembly and better understanding its mechanism of promoting plant growth.【Method】This study was based on a protoplast regeneration technique to obtain the regenerated strain, comparing the colony morphology and growth rate differences between the regenerated and original strains using the RNA polymerase second largest subunit gene (rpb2) and nuclear translation elongation factor subunit 1-α (tef) as conservative genes. This was then performed on PCR identification, taking 4', 6-diamidino-2-phenylindole (DAPI) fluorescence nucleus staining together to determine if the regenerated strain is homokaryotic.【Result】We provided three lines of evidence to confirm that we had obtained the homokaryotic strain. First, we found that the colony appearance and the growth rate differed considerably between the regenerated and original strains, even though they both lack the clamp connection. Second, DAPI fluorescence nucleus staining and confocal microscopy confirmed that the heterokaryotic hypha contains two nuclei. In contrast, each hyphal cell has a single nucleus in the regenerated strain at its immature stage, while growing into homokaryotic hyphae that contain two nuclei at maturity. Third, sanger sequencing of rpb2 and tef partial genes revealed that the original strain showed a degree of heterozygosity, but this was completely absent in the regenerated strain. 【Conclusion】 Collectively, these data suggest that the regenerated strain of C. hobsonii is homokaryotic. With accurate and stable genetic information, we can provide a material basis for obtaining genomic data of heterokaryon fungi.