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Glucosinolates (GSLs) are important secondary metabolites in Brassicales related to insect and disease resistance, flavor formation, and human health. Here, we determined the GSL profile with sinigrin as the predominant GSL in <i>Brassica nigra</i>. A total of 184 GSL biosynthetic genes (<i>BniGSLs</i>) were identified in <i>B. nigra</i> by a genome-wide search for orthologs of 82 of the 95 known GSL genes in <i>Arabidopsis thaliana</i>. Transcriptome data demonstrated that at least one <i>BniGSL</i> was highly expressed in stems and leaves at each step of the sinigrin synthesis pathway, which ensured the synthesis of a large amount of sinigrin in <i>B. nigra</i>. Among these key candidates of <i>BniGSLs</i>, the high expression of <i>BniMAM1-2</i>, <i>BniCYP79F1</i>, and <i>BniAOP2-1/2</i>, and the absence of <i>MAM3</i> and <i>AOP3</i>, may contribute remarkably to the synthesis and accumulation of sinigrin. In addition, the low expression of some key <i>BniGSLs</i> partially explains the low content of indolic and aromatic GSLs in <i>B. nigra</i>. This study provided a genetic explanation for the formation of the unique GSL profile with sinigrin as the main GSL in <i>B. nigra</i>. The results of this study will be valuable for further functional analysis of <i>BniGSLs</i> and genetic improvement of GSLs in <i>B. nigra</i> and other <i>Brassica</i> species.