Grafting has been widely used in orchard management because the rootstock can make the tree more tolerant to environmental stresses. Iron deficiency is one of the major limiting environmental factor in apple production worldwide. Systematic research has been made about iron-deficiency adaptive responses in the level of organs, cells, and subcells, whereas the interactions between Fe and other divalent cations in tissue level are little known. Synchrotron radiation X-ray fluorescence (SR-μXRF) was used to map the location of selected elements Fe, Zn, Mn, Ni, and Co in the longitudinal and latitudinal root samples of Malus xiaojinensis. Iron deficiency induced a significant increase in the relative contents of five micronutrients in epidermis and cortex. The ratio of element contents of roots under Fe-deficient condition and Fe-sufficient condition at same position increased obviously in the section of 1000- to 2000-μm distance from the root tip in xylem. Expression analysis of iron absorption- and transport-related genes in roots showed that MdNramp3 and MxCS1 increased significantly. These results indicated that iron deficiency promoted the long-distance transport of micronutrients in xylem, and MdNramp3 and MxCS1 might play an important role in this process. Importantly, this study directly provides visual divalent metals distribution in tissue level for an improved understanding of metal absorption process in apple rootstock.