Glucosinolates (GSLs) are significant and specialized metabolites found in Brassicas that have crucial roles in both human and plant defence. The present study investigated sinigrin, progoitrin and glucoerucin in Indian cauliflower genotypes using high-performance liquid chromatography (HPLC). For this, 37 genotypes of cauliflower from early (14), mid-early (6), mid-late (15) and late (2) maturity groups along with broccoli (two) and Sicilian purple (one) were evaluated in randomized block design during 2019–20 and 2020–21. Glucoerucin was predominant in most of the cauliflower genotypes (30), followed by sinigrin (5) and progoitrin (2). It was also prominent in broccoli genotypes. Progoitrin was the principal GLS in Sicilian Purple ‘PC-1 (2.430 μmol/g). In cauliflower, the glucoerucin, progoitrin and sinigrin were ranged from 0.067 to 7.248 μmol/g, 0.001 to 0.849 μmol/g and 0.001 to 3.310 μmol/g, respectively. Pusa Deepali (early), Pusa Sharad (mid-early) and Pusa Shukti (mid-late) were found to be ‘low progoitrin-high glucoerucin’ varieties in their respective groups. In the late group, Pusa Snowball Kt-25 had low progoitrin. Glucoerucin and sinigrin were highest in the mid-early group. Progoitrin was highest in genotypes harvested in the first fortnight of November and the second fortnight of February, whereas sinigrin and glucoerucin were maximum in the genotypes harvested during the second fortnight of November. The K-means clustering identified four clusters, and principal component analysis revealed two principal components. The information on three GLSs in Indian cauliflower will be useful for breeding varieties with desirable GSL profiles for public health and plant defence.