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The proteins in the starchy endosperm of wheat determine wheat quality and exhibit a quantitative gradient decreasing from the outer to inner endosperm. Here, we investigate how protein-rich sub-aleurone cells contribute to the protein content and gradient by studying three cultivars, each cultivated at three levels of nitrogen (N)-fertilization. The observed increased protein content with increased N-fertilization was cultivar-dependent. Image analysis showed that the underlying protein gradient could be described by a declining biexponential curve, with protein contents up to 32.0% in the sub-aleurone. Cultivars did not differ in protein content in the center of the cheeks and only differed in the outer endosperm when N-fertilization is applied. N-Fertilization resulted in relatively higher increases in protein content in the outer compared to inner endosperm. Hence, sub-aleurone cells could affect the classification of cultivars by baking quality. Cultivar selection and N-fertilization could furthermore be promising techniques to produce protein-rich miller’s bran.