The efficiency of fractionating cereal grains (e.g., dry corn milling) can be evaluated and monitored by quantifying the proportions of seed tissues in each of the recovered fractions. The quantities of individual tissues are typically estimated using indirect methods such as quantifying fiber or ash to indicate pericarp and tip cap contents, and oil to indicate germ content. More direct and reliable methods are possible with tissue-specific markers. We used two transgenic maize lines, one containing the fluorescent protein green fluorescent protein (GFP) variant S65T expressed in endosperm, and the other containing GFP expressed in germ to determine the fate of each tissue in the dry-milling fractionation process. The two lines were dry-milled to produce three fractions (bran-, endosperm-, and germ-rich fractions) and GFP fluorescence was quantified in each fraction to estimate the tissue composition. Using a simplified laboratory dry-milling procedure and our GFP-containing grain, we determined that the endosperm-rich fraction contained 4% germ tissue, the germ-rich fraction contained 28% germ, 20% endosperm, and 52% nonendosperm and nonembryo tissues, and the bran-rich fraction contained 44% endosperm, 13% germ, and 43% nonendosperm and nonembryo tissues. GFP-containing grain can be used to optimize existing fractionation methods and to develop improved processing strategies.