Shiitake breeding requires the procedures of mating of two different parental strains and selection of hybrid strains that have good traits for the mushroom production. In this study, we tested the possibility of the use of chromogenic plate-based assay for extracellular enzyme production in order to assess and find good bio-chemical properties-possessed hybrid strains that were generated from genetic cross of the monokaryotic strains derived from two different parental strains of Lentinula edodes Sanjo-101ho and Sanjo-108ho. We observed that there was difference in the ability of producing β-glucosidase, avicelase, CM-cellulase, amylase, pectinase, xylanase, and protease among the monokaryotic strains. We could also comparatively assess that the ability of the seven extracellular enzymes production in the hybrid strains depended on the mating combination of the monokaryotic strains. Our results demonstrate that the assessment method for extracellular enzyme production using chromogenic plate assay could be usefully applied to the assessment of the hybrid strains derived from the breeding procedure of L. edodes.