Most broccoli cultivars or accessions have green buds at an appropriate growth environment. They would develop red or purple buds at cold temperature, while some have green buds both at normal growth or cold temperature. In this study, through BSA-seq, fine mapping and transcriptome analyses, we identified a candidate gene ( F3’H) responsible for reducing the accumulation of anthocyanins in a mutant GS and HX-16 broccoli (DH line) ( Brassica oleracea L. var. italica), which would have caused green buds even at low temperature. A 43-bp deletion was detected in the CDS of the F3’H gene of HX-16 and mutant GS, which largely reduced the expression of F3’H and the accumulation of cyanidin and delphinidin in the mutant GS. The expression of F3’H was up-regulated at low temperature in the wild line PS.The detection of 43-bp deletion in the broccoli germplasm resources and other varietas in Brassica oleracea showed that there could be other factors for green buds at low temperature in Brassica oleracea. This study provides the critical genetic and molecular information for the molecular breeding of B. oleracea and illuminates the molecular basis of the low-temperature effect on buds’ color in broccoli.