The competitiveness of the mushroom industry can be increased by diversifying the domestic Lentinula edodes cultivar. Therefore, ‘Chungheung 1ho’ was cultivated by hybrid breeding method using strain ‘E140025,’ which was collected by the Chungheung Mushroom Farming Association. Regarding the morphological characteristics of the fruit, the cap of ‘Chungheung 1ho’ was smaller than that of the control cultivar, whereas, its stem was longer and thicker than that of the control cultivar. In addition, the weight of the individual fruits of the new cultivar was heavier than that of the control cultivar. The period for primordia formation was 24 days for the new cultivar, which was 16–20 days later than that of the control cultivar. Furthermore, it was found that the patterns of the PCR amplification band using six URP primers were different for ‘Chungheung 1ho’ than those of the mating strains.