In vitro fermentabilities of the oat flour digestion residues (ODR) from two commercial oat lines with 4.7 and 5.3% β-glucan and from two high-β-glucan experimental lines with 7.6 and 8.1% β-glucan were evaluated and compared with fermentations of lactulose, purified oat β-glucan (POBG), and purified oat starch (POS). Substrates were fermented by using an in vitro batch fermentation system under anaerobic conditions for 24 h. The progress of the fermentation was studied by following the change in pH of the fermentation medium, production of short-chain fatty acids (SCFA) and gases, and consumption of carbohydrates. The substrate from the flour with the greatest amount of β-glucan tended to have the greatest pH decline and the greatest total SCFA production. A significant correlation occurred between gas production and SCFA formation (R 2 = 0.89−0.99). Acetate was produced in the greatest amounts by all of the substrates except POBG, by which butyrate was produced in the greatest amount. More propionate and butyrate, but less acetate, were produced from high-β-glucan ODR. With the given fermentation conditions, >80% of the total carbohydrate was depleted by the bacteria after 24 h. Glucose was the most rapidly consumed carbohydrate among other available monosaccharides in the fermentation medium. Overall, the high-β-glucan experimental lines provided the best conditions for optimal in vitro gut fermentations.