Light transmission through wheat (T. aestivum L.) grain longitudinal cross sections of different thickness was used to study the endosperm microstructure and was shown to strictly follow a Beer-Lambert law allowing a non ambiguous quantification of the endosperm vitreousness. Therefore similar samples obtained from near-isogenic lines differing by hardness and grown in two distinct environments affecting their vitreousness were analyzed and confirmed the relationship between light transmission and the endosperm microstructure. In each sample, moreover analysis of light transmission within the different grain parts highlighted the greater compactness of the central endosperm cheeks in comparison with the distal and the proximal regions. These results helps a better understanding of the endosperm microstructure.