This study investigated the properties of starch isolated from Betaone barley (high β-glucan content of 11.32%), waxy-type Hwanggeumchal barley, and non-waxy type Hyeyang barley using water or 0.2% NaOH. Starch isolated with NaOH contained 4.79% damaged starch, 0.17% protein, 0.01% β-glucan, and 0.20% crude fat, while starch isolated with water contained 2.95%, 1.91%, 0.10%, and 0.31%, respectively. The starch granule were oval in shape, with particle sizes ordered as follows: Betaone (4.9 μm), Hwanggeumchal (8.9 μm), and Hyeyang (13.6 μm). Thermal properties revealed, that gelatinization onset, peak, and end temperatures increased with particle size. NaOH isolation was found to be more effective than water isolation in removing proteins, but resulted in higher levels of damaged starch and partially disrupted the crystalline structure.