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Allopolyploid Brassica juncea is particularly enriched in sinigrin, a kind of 3C aliphatic glucosinolates (GSLs), giving rise to characteristic taste after picking. However, the molecular mechanism underlying 3C aliphatic GSLs biosynthesis in this species remains unknown. In this study, we genome-widely identified GSLs metabolic genes, indicating different evolutionary rate of GSLs metabolic genes between subgenomes of B. juncea. Eight methythioalkylmalate synthase (MAMs) homologs were identified from B. juncea, in which six MAM1s were located in chloroplast and the other two were not detected with any expression. Furthermore, BjMAM1-4, BjMAM1-5, and BjMAM1-6 displayed higher expression levels in leaves than other tissues. Silenced expression analysis revealed that BjMAM1-4 and BjMAM1-6 function in 3C and 4C aliphatic GSLs accumulation. The specificity of the substrate selection for the second cycle reaction is much lower than that of the first cycle, suggesting these genes may preferentially catalyze 3C aliphatic GSLs biosynthesis. Our study provides insights into the molecular mechanism underlying the accumulation of 3C aliphatic GSLs, thereby facilitating the manipulation of aliphatic GSLs content in B. juncea.