The Grifola frondosa cultivar KMCC03118 was used to isolate monokaryotic strains via spore separation, resulting in the successful crossbreeding of strains KMCC03118-11 and KMCC03118-23, which produced F1 hybrids. These F1 hybrids were then further crossed with various monokaryotic strains to generate F2 progeny. In evaluating the effects of different medium compositions on fruit body development, it was found that a substrate consisting of wheat bran and dried sawdust, with a carbon-to-nitrogen (C/N) ratio of 66-68, provided the most favorable conditions for mycelial growth. Among the strains tested, KMCC03137 and GF-18-50 demonstrated superior characteristics, including a larger fruit body diameter, thicker pileus, and greater stipe thickness, with the highest productivity observed at 143.6 ± 13.3 g and 144.7 ± 15.2 g, respectively. Furthermore, the color of the caps (L: 29.7 ± 7.1, a: 2.6 ± 0.7, b: 8.2 ± 1.8) remained consistent, indicating stable high-quality production. Based on these results, the optimal substrate composition for enhancing both the quality and productivity of the fruit bodies was determined to be 42% Quercus sp. sawdust, 42% Quercus sp. fermented sawdust, 6% wheat bran, and 10% dried tofu residue. This study provides a crucial foundation for the commercial cultivation and breeding improvement of Grifola frondosa, offering valuable insights into its genetic enhancement, and providing essential data for future research aimed at increasing the species' genetic diversity and productivity.