Five different barley cultivars, including covered and naked samples containing low, normal, or high-amylose starches were fractionated by weighing, pneumatic classification, sieving, or sorting on a specific gravity table, and analyzed for content of starch, protein, ash, and β-glucan. For ash content, almost no variation could be found between different fractions. Protein content was minimum in the intermediate fractions for all cultivars when sorted by weighing. For the other fractionation methods, the differences in protein content were small. A tendency for decreasing content of starch with increasing grain mass and size could be seen when fractionating grains by weighing and sieving, respectively. The clearest trend was seen in differences in β-glucan content for all cultivars and all methods used. The main interpretation of our results is, however, that the chemical composition within the cultivars studied is very similar for all fractions, and that the differences between the unfractionated barley samples are larger.