Domestic wheat flour is generally darker than imported flours, and wheat researchers would like to elucidate the cause of darkness in wheat. A high-throughput endosperm colorimetry system has been established to evaluate flour color without an effect of milling fraction. Using the system, we investigated whether the endosperm brightness (L^* value) was associated with the protein and iron contents. We found that as the endosperm L^* value rose, the protein and iron contents declined. However, among samples with similar protein contents, there was no correlation between endosperm L^* value and iron content. Therefore, we conclude that iron content is not a main cause of endosperm darkness. It is possible that the reported relationship between flour darkness and its iron content is caused by bran included in the flour and/or by a positive correlation of protein and iron content.