Deciphering how cellular iron (Fe) pools are formed, where they are localized, and which ones are remobilized represents an important challenge to better understand Fe homeostasis. The recent development of imaging techniques, adapted to plants, has helped gain insight into these events. We have analyzed the localization of Fe during embryo development in Arabidopsis (Arabidopsis thaliana) with an improved histochemical staining based on Perls coloration intensified by a second reaction with diaminobenzidine and hydrogen peroxide. The procedure, quick to set up and specific for Fe, was applied directly on histological sections, which dramatically increased its subcellular resolution. We have thus unambiguously shown that in dry seeds Fe is primarily stored in the endodermis cell layer, within the vacuoles, from which it is remobilized during germination. In the vit1-1 mutant, in which the Fe pattern is disturbed, Fe is stored in vacuoles of cortex cells of the hypocotyl/radicle axis and in a single subepidermal cell layer in the cotyledons. During the early stages of embryo development, Fe is evenly distributed in the cells of both wild-type and vit1-1 mutants. Fe eventually accumulates in endodermal cells as the vascular system develops, a process that is impaired in vit1-1. Our results have uncovered a new role for the endodermis in Fe storage in the embryo and have established that the Perls/diaminobenzidine staining is a method of choice to detect Fe in plant tissues and cells.