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Wheat gluten properties can be improved by the application of nitrogen. This study investigates the effects of nitrogen application in the booting stage on glutenin polymerization during grain-filling and structural&#8722;thermal properties of gluten based on the high-molecular-weight glutenin subunits (HMW-GSs) using near-isogenic lines (<i>Glu-1Da</i> and <i>Glu-1Dd</i>). The nitrogen rate experiment included rates of 0, 60, 90, and 120 kg N ha^&#8722;1 applied with three replicates. Nitrogen significantly improved the grain quality traits (wet gluten contents, Zeleny sedimentation values, and maximum resistance) and dough strength (dough development time, dough stability time, and protein weakening), especially in wheat with the <i>Glu-1Da</i> allele. Nitrogen increased the protein composition contents, proportions of glutenins and HMW-GSs, and disulfide bond concentration in the flours of <i>Glu-1Da</i> and <i>Glu-1Dd</i>, and accelerated the polymerization of glutenins (appearing as glutenin macropolymer) during grain-filling, where nitrogen enhanced the accumulation and polymerization of glutenins more for line containing <i>Glu-1Da</i> than <i>Glu-1Dd</i>. The &#946;-sheets, &#945;-helix/&#946;-sheet ratio, microstructures, and thermal stability were also improved to a greater degree by nitrogen for gluten with <i>Glu-1Da</i> compared to <i>Glu-1Dd</i>. Nitrogen treatment was highly effective at improving the gluten structural‒thermal properties of wheat in the booting stage, especially with inferior glutenin subunits.