Abstract: Wheat grain quality had attracted a significant concern with increasing grain yield in recent years and different cultivation measures had drastically influenced wheat grain quality. To explore the effects of nitrogen fertilizer dose and row spacing on grain quality of strong and medium gluten wheat varieties and differences between two wheat types, an experiment was conducted at the experimental station of the Institute of Crop Science of Chinese Academy of Agricultural Sciences in 2013–2014. Strong gluten wheat variety ‘Jimai 20’ and medium gluten wheat variety ‘Zhongmai 8’ were used in a three-factor complete split-plot design. The main plot consisted of three levels of nitrogen fertilization rates — 150 kg(N)·hm^-2, 210 kg(N)·hm^-2 and 270 kg(N)·hm^-2. Then the first sub-plot consisted of two levels of row spacing in 12 cm and 20 cm, and the second sub-plot of two levels of wheat varieties (‘Jimai 20’ and ‘Zhongmai 8’). The results showed that protein accumulation after anthesis and protein content at maturity of ‘Jimai 20’ and ‘Zhongmai 8’ wheat varieties improved significantly with increasing nitrogen fertilizer dose and row spacing. The effect of nitrogen fertilizer on protein accumulation after anthesis was most notable in low N level treatment. A significant difference existed between the two different gluten wheat varieties with increasing row spacing in terms of grain protein accumulation during late grain-filling stage. Under 20 cm row spacing, 210 kg·hm^-2 N was more favorable in terms of improved kernel hardness, flour yield, wet gluten content, sedimentation volume and farinograph parameter of ‘Jimai 20’ wheat variety. Then with nitrogen fertilizer dose of 270 kg·hm^-2, there was high processing quality of ‘Zhongmai 8’ wheat variety. With increasing nitrogen fertilizer beyond this dose, bread volume and bread score significantly increased in‘Jimai 20’ and ‘Zhongmai 8’ wheat varieties, with decreasing volume weight of the two wheat varieties. In addition, increasing row spacing notably improved the processing quality of ‘Jimai 20’ and ‘Zhongmai 8’ under nitrogen fertilizer dose greater than 150 kg·hm^-2. It then implied that the processing quality of different gluten wheat varieties was better at row spacing of 20 cm than at row spacing of 12 cm. Grain quality of winter wheat could be improved under better nitrogen application and row spacing. The most appropriate treatments for strong gluten wheat (‘Jimai 20’) and medium gluten wheat (‘Zhongmai 8’) were respectively 20 cm row spacing with 210 kg·hm^-2 nitrogen dose and 20 cm row spacing with 270 kg·hm^-2 nitrogen dose. The experimental analysis showed that increasing nitrogen fertilizer application and row spacing was beneficially improved grain quality of strong gluten and medium gluten winter wheat varieties.