The distribution of a fraction of soluble dietary fibre (1,3)(1,4)-β-D-glucans was depicted in selected Polish oat grains (Avena sativa L.). Localization of β-glucans within the grains were visualised by the light microscopy with Calcofluor white as fluorescence agent. Content of β-glucans varied in samples in range from 3.08% d.m. to 5.04% d.m. Analysis of distribution of (1,3)(1,4)-β-D-glucans showed that localization of β-glucans varied between the different cultivars and lines. It was demonstrated that the total content of (1,3)(1,4)-β-D-glucans in oat kernels had an effect on their distribution. All the lines and cultivars tested displayed the greatest accumulation of (1,3)(1,4)-β-D-glucans in the cells of the subaleurone layer. With increase in the levels of β-glucans in high-glucan oat cultivars and lines, a tendency was observed towards their greater accumulation in the central parts of the kernel. It makes oat grain particularly suitable for the production of wholemeal foods. It is important to not only focus on increasing the amount of the β-glucan, but also investigate how the molecule is distributed in the seed. It was also demonstrated that cultivar Dukat is characterised by an especially valuable triple aleurone layer, what makes this cultivar predestined for further breeding studies as an extremely valuable carrier of genetic information.