<p>High dosages and split application of nitrogen (N) fertilizers are common practices for yield gains in wheat globally. N remobilization to developing grain depends on the N reserve pool of flag leaf. Studies on changes in flag leaf N assimilation with split N fertilization and N foliar spray are limited. The present research was aimed to assess the role of split N fertilization on flag leaf N assimilation, productivity and quality of wheat. The wheat cultivars viz. <i>Unnat</i> PBW 550, BWL 7504, BWL 7509 were the main plot treatments with soil N fertilization and foliar N spray the subplot treatments. A significant increase in the activities of N assimilation enzymes of flag leaves i.e. glutamine synthetase (GS), glutamate synthase (GOGAT) and glutamate pyruvate transaminase (GPT) was elicited with N application compared to the control (No N applied). Further acceleration in activities of GS (10.2–15.4%), GOGAT (15.5–20.6%) and GPT (2.3–14.3%) was more prominent in plants receiving higher N dosage of N₁₅₀ compared to N₁₂₀ application. The levels of free amino acids, soluble proteins, and soluble sugars showed a significant rise of 4.0%, 13%, and 12.8%, respectively, in plants where N₁₂₀ was applied in 4 splits compared to 3 splits alone, but no such response occurred at the higher N dose (N150) on a pooled basis. There was genotype specific behaviour in N metabolism enzymes where <i>Unnat</i> PBW550 and BWL 7509 performed better in enzymatic activities and had higher protein and free amino acid levels in flag leaves than BWL 7504. Significant increments in grain yields occurred with N fertilization, particularly in N₁₅₀ applied plants irrespective of 3 splits or 4 splits application. This increment was also significant in yield attributes i.e. effective tillers, number of grains per spike and 1000 grain weight, where a 2-fold yield increase occurred in plants with N application compared to the control (with no N application). Grain quality traits viz. grain protein content, gluten content and grain N removal exhibited a significant increase with N fertilization with N₁₅₀ dose (in 4 splits treatment) resulted in superior grain quality and higher grain N concentration than N₁₂₀ (3 or 4 splits). Foliar spray of urea/nano urea with soil N application in 3 splits were similar in productivity traits and quality attributes to soil N application in 4 splits irrespective of N dose. Correlation analysis revealed a strong positive association of activities of nitrogen assimilation enzymes and grain yield, grain protein content and grain N concentration. The genotype Unnat PBW 550 and BWL 7509 were superior in quality traits and yield performance over BWL 7504. It is concluded that four split applications of N₁₅₀ dose resulted in better N assimilation and N acquisition in wheat grain; thereby improve grain quality.</p> Graphical Abstract <p></p>