Many wheat species and cultivars, independent of genetic markers of hardness, can produce grain with a vitreous, mealy or mixed appearance. This study analyzed selected chemical and physical differences between kernels with a vitreous and mealy appearance, hand-picked from grain of four winter wheat cultivars cultivated in Poland. Separated fractions were examined for protein content and composition, friabilin presence, carotenoids and total phenolic compounds content, specific kernel density, hardness, as well as kernel surface color. It was found that the ratio of vitreous kernels in the cultivars ranged from 39.18% to 76.28%. Vitreous kernels were darker, slightly heavier and harder than mealy kernels. Additionally, these kernels were more abundant in proteins (an average increase of 2.13%, with variation among cultivars from 0.71% to 2.89%). This type of kernels was also richer in phenolic compounds (on average by 4.02%) and less abundant in carotenoids (on average by 4.53%). Mealy (softer) kernels fractured to a finer flour.