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<em>CYP83A1</em> and <em>CYP83B1</em> are two key synthesis genes in the glucosinolate biosynthesis pathway. <em>CYP83A1</em> mainly metabolizes the aliphatic oximes to form aliphatic glucosinolate and <em>CYP83B1</em> mostly catalyzes aromatic oximes to synthesis corresponding substrates for aromatic and indolic glucosinolates. In this study, two <em>CYP83A1</em> genes named <em>BcCYP83A1-1</em> (JQ289997), <em>BcCYP83A1-2</em> (JQ289996) respectively and one <em>CYP83B1</em> (<em>BcCYP83B1</em>, HM347235) gene were cloned from the leaves of pak choi (<em>Brassica rapa</em> L. ssp. <em>chinensis </em>var. <em>communis </em>(N. Tsen & S.H. Lee) Hanelt) “Hangzhou You Dong Er” cultivar. Their ORFs were 1506, 1509 and 1500 bp in length, encoding 501, 502 and 499 amino acids, respectively. The predicted amino acid sequences of <em>CYP83A1-1</em>, <em>CYP83A1-2 </em>and <em>CYP83B1</em> shared high sequence identity of 87.65, 86.48 and 95.59% to the corresponding ones in <em>Arabidopsis</em>, and 98.80, 98.61 and 98.80% to the corresponding ones in <em>Brassica pekinensis </em>(Chinese cabbage), respectively. Quantitative real-time PCR analysis indicated that both <em>CYP83A1</em> and <em>CYP83B1</em> expressed in roots, leaves and petioles of pak choi, while the transcript abundances of <em>CYP83A1 </em>were higher in leaves than in petioles and roots, whereas <em>CYP83B1 </em>showed higher abundances in roots. The expression levels of glucosinolate biosynthetic genes were consistent with the glucosinolate profile accumulation in shoots of seven cultivars and three organs. The isolation and characterization of the glucosinolate synthesis genes in pak choi would promote the way for further development of agronomic traits via genetic engineering.