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Excessive application of nitrogen fertilizer in the swollen-stem mustard cultivation leads to a series of environmental and quality issues. It was reported that reducing nitrogen fertilizer with biochar could increase crop yield and reduce environmental risks. However, the effect of nitrogen reduction combined with biochar application on the flavor substances was rarely reported. Thus, two genetic stem mustard varieties (Yx: Yong'an xiaoye and Fz: Fuza No. 2), and four N treatments (control: 0 N kg/ha with biochar; N150: 150 N kg/ha with biochar, N300: 300 N kg/ha with biochar, and N450: 450 N kg/ha) were chosen to study the effects of nitrogen reduction combined with biochar on the flavor substance content of mustard stem, and N balance. The results showed that the residual soil inorganic N in N300 was lower by 37% than that in N450 (156.5 kg/ha) in Fz mustard soil, and lower by 33% in N150 than in N450 (163.1 kg/ha) in Yx mustard. The highest biomass of stem mustard tumors of Fz (35.4 × 103 kg/ha) and Yx (35.7 × 103 kg/ha) was in N300. The content of umami amino acids, sweet amino acids, and bitter amino acids of Yx and Fz stem was the highest in N450, and N300, respectively. After comprehensive consideration, the Fz was recommended to be planted in the Three Gorges Reservoir Area with N300. [ABSTRACT FROM AUTHOR]