β-Glucans are soluble dietary fibers predominantly found in cereals and are characterized by β-(1→3)/(1→4) glycosidic linkages, which define their unique structural and functional properties. Barley and oat β-glucans have received approved health claims from regulatory authorities due to their recognized roles in the prevention of metabolic disorders. In this study, the rheological, structural, and functional properties of powdered, concentrated β-glucans from barley and oat were investigated. Rheological characterization included dynamic viscoelastic measurements and steady shear viscosity at 10% and 15% concentrations using an AR-G2 rheometer. Particle size distribution was determined using a Mastersizer analyzer. Structural features were evaluated by FT-IR spectroscopy, size-exclusion chromatography, and differential scanning calorimetry (DSC) to determine thermodynamic parameters. β-Glucan quantification was performed using the Megazyme β-glucan assay kit, and functional properties were assessed by measuring water absorption capacity (WAC) and oil absorption capacity (OAC). Overall, both barley and oat β-glucan powders exhibited similar rheological behavior, with higher shear stress required under increasing shear rates, and storage modulus (G′) exceeding loss modulus (G″), indicating predominantly elastic or solid-like gel characteristics. Oat β-glucan displayed a larger median particle size (d₅₀ = 532 µm) compared to barley (d₅₀ = 248 µm), whereas barley β-glucan exhibited higher molecular weight and greater β-glucan content. The FT-IR spectra of both samples showed similar structural patterns, with the highest absorption peak between 1000–1200 cm⁻¹ corresponding to C–O–C stretching vibrations. Barley β-glucan showed overall higher transmittance values, suggesting subtle structural differences compared to oat β-glucan. The thermal analysis revealed slightly higher, though not significantly different, onset, peak, and endset temperatures, as well as a higher gelatinization enthalpy for barley β-glucan. Regarding WAC, both oat and barley showed similar water absorption capacities, whereas barley exhibited a higher oil absorption capacity (OAC). These results demonstrate that while oat and barley β-glucans share similar structural motifs, variations in molecular weight, particle size, and rheological behavior may influence their functional performance in food and nutraceutical applications.