Discolored grain is one of the most important types of damage in two-rowed hulled barley. We attempted to elucidate the occurrence factors in discolored grain and to establish an evaluation method. To reveal the occurrence factors in discolored grain, three color values (L* value: brightness, a* value: redness and b* value: yellowness) of discolored grain were investigated to see whether they correlated with the amount of precipitation during the maturation period. The hue of grains showed no correlation with the amount of precipitation from 4 to 2 weeks (yellow ripe stage) before the mature stage, whereas the L* value and b* value of grains correlated negatively with the amount of precipitation during from the yellow ripe stage to the mature stage. The differences in the L* value and b* value of grains with high humidity treatment for 4 days and no treatment using detached spikes in the yellow ripe stage in 31 varieties correlated positively with the degree of discoloration of grain under natural conditions. These results showed that discolored grain developed by exposure to rainfall from the yellow ripe stage to mature stage, could be evaluated by high humidity treatment, and varieties tolerant to grain discoloration could be selected using this treatment.