Nitrogen (N) is crucial for wheat yield and grain quality, both influenced by cultivar, soil, climate, and management. Environmental variation makes quality hard to predict across sites and years. This study assessed winter wheat grain quality under organic and mineral fertilization at two Czech sites. Mineral N fertilization consistently gave the highest yield and protein content, while organic fertilizers improved yield but often failed to meet food-grade standards. Temperature during grain filling strongly affected bulk density and interannual variability. A two-year field trial compared six treatments: Control, Sewage Sludge (SS), Farmyard Manure (FYM), Mineral N, NPK, and Mineral N + straw (N+ST). Grain nitrogen was measured by the Kjeldahl method (VAPODEST 50s), after homogenization with an SM 100 mill. Results were statistically analyzed over two years using ANOVA and Tukey’s test in STATISTICA 12.0.