Understanding the accumulation and distribution of essential nutrients in cereals is of primary importance for improving the nutritional quality of this staple food. While recent studies have improved the understanding of micronutrient loading into the barley grain, a detailed characterization of the distribution of micronutrients within the grain is still lacking. High-definition synchrotron X-ray fluorescence was used to investigate the distribution and association of essential elements in barley grain at the micro scale. Micronutrient distribution within the scutellum and the embryo was shown to be highly variable between elements in relation to various morphological features. In the rest of the grain, the distribution of some elements such as Cu and Zn was not limited to the aleurone layer but extended into the endosperm. This pattern of distribution was less marked in the case of Fe and, in particular, Mn. A significant difference in element distribution was also found between the ventral and dorsal part of the grains. The correlation between the elements was not consistent between and within tissues, indicating that the transport and storage of elements is highly regulated. The complexity of the spatial distribution and associations has important implications for improving the nutritional content of cereal crops such as barley.