Two Tunisian barley varieties, Martin and Rihane, differing by their degrees of adaptation to the environmental conditions and grain yield potential, were grown under different levels of nitrogen (N) fertilisation. The effect of nitrogen supply on the hordein components (expressed in mg per albumen) was determined by Nu-PAGE electrophoresis system using the MOBS running buffer and densitometric analysis. Increasing nitrogen fertilisation increased the protein content by increasing the amount of C-hordeins. The sub-fraction BIII of high molecular weight B-hordeins appeared, in the present study, as the best criterion for distinguishing between the two barley varieties genetic adaptation, and its quantification could be recommended for the understanding of the quality properties of the barley response to different environmental conditions particularly nitrogen fertilisation which appears to be the main origin of differences in the protein aggregation mode of different B-hordein sulphur-rich sub-fractions.