Lentinula edodes have been cultivated extensively not only in South Korea but also in other Asian countries. In terms of taste and nutrition, it is a valuable mushroom that is comparable to other mushrooms. L. edodes contains various physiologically active compounds that promote human health. One compound, ergothioneine, exerts a powerful antioxidant function that inhibits cellular senescence. As it is not biosynthesized by plants or animals, L. edodes is very important for ergothioneine supply. The L. edodes cultivars Bambithyang (NIFoS 3404) and Sansanhyang (NIFoS 3420) of the National Institute of Forest Science (NIFoS) had higher ergothioneine content than the other cultivars, and 11 strains were obtained using mono-mono hybridization between them. The strains were inoculated into sawdust media to obtain fruiting bodies, and the ergothioneine content of each hybrid strain was confirmed using high-performance liquid chromatography (HPLC) analysis. Among the 11 strains, NIFoS 5108 had a higher ergothioneine content than the parental strains, Bambithyang (NIFoS 3404) and Sansanhyang (NIFoS 3420). This study revealed that it is possible to develop cultivars with improved specific functions using mono-mono hybridization.