Genetic engineering of crop plants relies on the development of efficient methods for the regeneration of viable shoots from cultured tissues. The objective of the present study was to develop a protocol for efficient shoot and plant regeneration from seedling explants of commercial cauliflower (B. oleracea var. botrytis) genotypes and to compare the regeneration capacity of the most commonly used explants: cotyledon, hypocotyl, and root. A combination of growth hormones including 6-benzylaminopurine, 1-naphthylacetic acid, and gibberellic acid was used in the MS-based medium, and factors influencing regeneration of shoots were investigated. Using the protocol described here, shoots from hypocotyl, cotyledon, and root explants of all the 11 genotypes tested were able to be regenerated. Root and hypocotyl explants produced more callus than cotyledon explants and also were more responsive to shoot regeneration, as a high percentage (>71% and >80% for hypocotyl and roots, respectively) of shoot initiation from these explants was observed. In addition, root and hypocotyl explants also produced more shoots per explant than cotyledon explants. The in vitro regenerated shoots were successfully rooted and acclimatised to glasshouse conditions. This study shows that seedling explants of cauliflower are amenable to multiple shoot formation with high regeneration frequencies, and could be used for genetic transformation experiments.