Kernel hardness is an important trait influencing postharvest handling, processing, and food product quality in cereal grains. Though well-characterized in wheat, the basis of kernel hardness is still not completely understood in barley. Kernels of 959 barley breeding lines were evaluated for hardness using the Single Kernel Characterization System (SKCS). Barley lines exhibited a broad range of hardness index (HI) values at 30.1-91.9. Distribution of kernel diameter and weight were 1.7-2.9 mm and 24.9-53.7 mg, respectively. The proportion of hull was 10.2-20.7%. From the 959 breeding lines, 10 hulled spring barley lines differing in HI values (30.1-91.2) were selected to study the associations of HI with proportion of hull, kernel weight, diameter, vitreousness, protein, β-glucan, and amylose content. Vitreousness, evaluated visually using a light box, showed a clear distinction between hard and soft kernels. Hard kernels appeared translucent, while soft kernels appeared opaque when illuminated from below on the light box. Kernel brightness (L*), determined as an indicator of kernel vitreousness, showed a significant negative correlation (r = -0.83, P < 0.01) with HI. Protein, β-glucan, amylose content, proportion of hull, kernel weight, and diameter did not show any significant association with HI.