Breeding barley with a high content of dietary fiber, namely β-glucan, is required because β-glucan is attracting attention for its health functionalities such as suppression of postprandial blood glucose elevation. We analyzed the relationship between the β-glucan content and the starch synthase IIIa (ssIIIa) marker type, which is located near the region affecting the high amylose content amo1 mutation. The ssIIIa marker types were detected, and the β-glucan content, SKCS grain hardness, steely grain rate, thousand-grain weight, and hollow grain rate were measured for each of the 94 waxy lines (F5) derived from a cross between ‘Hokuriku 54’ (later ‘Yukihanarokujo’) and the amo1 waxy variety ‘YonRkei3755’, and for each of the 165 waxy lines (F5) derived from a cross between ‘YonRkei3755’ and the waxy variety ‘Haneumamochi’. As a result, there were significant differences in the β-glucan content, SKCS grain hardness, and steely grain rate depending on the ssIIIa marker type in each post-interbreeding line. The β-glucan content was about 1.6 times higher, the SKCS grain hardness was 23 points higher, and the steely grain rate was 7% higher in the ‘YonRkei3755’ ssIIIa type than that of the wild type. The β-glucan content was highly correlated with the SKCS grain hardness of r = 0.75 to 0.84 in the breeding line derived from both crosses. These results suggest that selection based on the ssIIIa marker type and/or the SKCS grain hardness are effective in breeding amo1 varieties with a high content of β-glucan.