A field experiment was conducted in 2018-2020 to investigate the effect of two nitrogen application rates (N 0 = 0 kg ha −1 (control), N 90 = 90 kg ha −1 , and N 180 = 180 kg ha −1 ) on morphological and physiological behaviours of two new rapeseed varieties; V 1 = JYJS01 (semi-dwarf variety) and V 2 = CY36 (tall variety). Our results indicated that N application enhanced the plant growth and seed protein content in comparison to control. Contrastingly, compared with control, sugar contents, i.e. sucrose and reducing sugars, decreased significantly in response to an increase in N. Seed oil content also decreased by 8.5% and 5.5%, 6.7% and 3.9% against N 180 in V 1 and V 2 during 2019 and 2020, respectively. Furthermore, higher nitrogen application rates decreased seed fatty acid proportions by decreasing the sugar availability for fatty acid biosynthesis. Our results demonstrated the highest seed yield (9.4 g plant −1 in 2018/19 and 9.5 g plant −1 in 2019/20) in V 2 against N 180 , and the lowest seed yield (3.2 g plant −1 in 2018/19 and 3.4 g plant −1 in 2019/20) in V 1 at control. These findings imply that a high nitrogen application rate had increased the yield. At the same time, the carbohydrates translocation from stems to seed for fatty acid biosynthesis decreased, which played their significant role in seed physiology for fatty acid metabolisms, ultimately affected the seed quality. However, there is still a dire need to evaluate how nitrogen application affect the carbohydrate availability for fatty acid metabolism in the seed of new winter rapeseed varieties.