Kale (Brassica oleracea var. acephala) is one of the most frequently consumed leafy vegetables globally, as it contains numerous nutrients; essential amino acids, phenolics, vitamins, and minerals, and is particularly rich in glucosinolates. However, the differences in the biosynthesis of glucosinolates and related gene expression among kale cultivars has been poorly reported. In this study, we investigated glucosinolates profile and content in three different kale cultivars, including green (‘Man-Choo’ and ‘Mat-Jjang’) and red kale (‘Red-Curled’) cultivars grown in a vertical farm, using transcriptomic and metabolomic analyses. The growth and development of the green kale cultivars were higher than those of the red kale cultivar at 6 weeks after cultivation. High-performance liquid chromatography (HPLC) analysis revealed five glucosinolates in the ‘Man-Choo’ cultivar, and four glucosinolates in the ‘Mat-Jjang’ and ‘Red-Curled’ cultivars. Glucobrassicin was the most predominant glucosinolate followed by gluconastrutiin in all the cultivars. In contrast, other glucosinolates were highly dependent to the genotypes. The highest total glucosinolates was found in the ‘Red-Curled’ cultivar, which followed by ‘Man-Choo’ and ‘Mat-Jjang’. Based on transcriptome analysis, eight genes were involved in glucosinolate biosynthesis. The overall results suggest that the glucosinolate content and accumulation patterns differ according to the kale cultivar and differential expression of glucosinolate biosynthetic genes.