Localization of iron (Fe), zinc (Zn), and protein was studied in a set of spelt (Triticum aestivum ssp. spelta) genotypes selected for low (i.e. ~12 %) or high (i.e. ~25 %) grain protein concentration. Following instrumental analysis for Fe, Zn and protein, spelt seeds were longitudinally excised and stained with specific dyes for assessment of Fe, Zn, and protein localization. For Fe and Zn staining, pre-defined methods with Perls’ Prussian blue and dithizone were applied whereas protein staining was performed by a modified Bradford reagent. Following staining, seed surfaces were examined by light reflectance microscopy and photographed to (i) examine the localization of Fe, Zn and protein and (ii) visually compare the variations in color intensity with the concentration data obtained by instrumental analysis of seeds with contrasting protein concentration. The applied staining method revealed that Fe localization was limited to scutellum and aleuron; however Zn and protein was localized in the whole germ and the aleuron as well. It was concluded that the staining methods applied for protein and Fe localization can also be used for mass screening of spelt genotypes for high seed protein and Fe concentration. The role of seed proteins as a sink for Fe and Zn in the spelt seed is discussed.