Grain mass (mg) and grain nitrogen concentration (%) were determined in 3,278 individual grains from eight cultivars with reference to the position of the grain on the head (spikelet number and floret number). Selected grains were removed from certain heads at anthesis. Grain nitrogen content (mg) was determined as the product of grain nitrogen concentration and grain mass. Grain mass and starch B-granule content were determined in 3,030 grains from a further 12 cultivars with reference to grain position, and selected grains were removed from certain heads at anthesis. Grains from distal florets were always smaller and had lower B-granule contents, nitrogen contents, and nitrogen concentrations than those from the two proximal florets on each spikelet, which were not significantly different from each other. Grains in the basal two spikelets of the head were smaller with lower nitrogen contents and higher B-granule contents than those in most of the head. Their nitrogen concentration, however, did not differ from that in the rest of the head. All four traits declined in the grains in the top four to five spikelets of the head. The differences in grain mass, nitrogen, and B-granule content between florets within spikelets and between spikelets within the head varied with cultivar. Grains on treated heads were larger, higher in nitrogen and slightly lower in B-granule content than those on untreated heads. The effects of floret and spikelet on grain mass and nitrogen were not significantly changed by the treatments. Grains from florets 1 and 2, excluding those from the bottom three and top five spikelets, therefore represented a particularly uniform population for grain mass, protein nitrogen and starch granule composition. Genetic analyses based on samples of these grains will be more robust and repeatable than those based on unselected samples. Implications for improving seedling vigor, plant yield, and grain protein concentration in breeding programs are discussed.