Two naked waxy barley cultivars, Bz 489-30 and Prowashonupana were analysed in order to study the chemical composition and the microstructure. Prowashonupana contained more protein, fat and ash, and less starch than Bz 489-30. The content of mixed-linked β-glucan was almost three times higher in Prowashonupana (14·9%) than in Bz 489-30 (5·6%), but extractability at 40 °C was much lower. Microscopy studies showed that kernels and endosperm cells of Prowashonupana were irregular, and that cell walls were thicker with a higher content of mixed-linked β-glucan than in Bz 489-30. In both barleys the sub-aleurone layer contained black starch granules, indicating a higher amylose content in this structure compared to the rest of the kernel. Starch was isolated from both barleys using two different procedures and analysed for amylose content. The amylose content was higher, while volume % of A-granules was lower and mean diameter of A-granules was smaller in starch isolated from Prowashonupana than from Bz 489-30. Both composition and yield of starch were however influenced by the isolation procedure used. The procedure which gave higher starch yield resulted in a higher amylose content, probably because of greater recovery of starch from outer parts of the kernel with a higher amylose content.