Seed tissues (endosperm, embryo, and pericarp) are often separated into tissue-enriched fractions by wet- or dry-milling methods for use in food, feed, and industrial products. Seed tissue markers that are sensitive and readily quantifiable would be useful to optimize fractionation processes. To meet this need for tissue markers, we set out to produce and characterize different transgenic maize lines, each containing green fluorescent protein (GFP) in either endosperm or embryo. We examined mRNA transcripts using expressed sequence tag (EST) profiles of several major seed proteins and selected several with strong seed tissue preferences. Stably transformed maize lines were produced, and visual observation of fluorescence confirmed the presence of GFP in the desired tissues. To establish the utility of this grain for evaluating the effectiveness or separation efficiencies of fractionation processes, transgenic kernels were hand-dissected into pericarp, endosperm, and embryo fractions and the GFP concentration in each fraction was determined. The GFP distribution between fractions of each transgenic event was calculated from GFP concentration and mass balance, which enabled the determination of GFP yield based on the hand-dissection fractionation data and the amount of tissue contamination in each fraction. Our transgenic lines exhibited strong tissue preference for either embryo or endosperm. These lines should be useful for assessing separation efficiencies in maize fractionation processes.