Radish (Raphanus sativus) plants exhibit varied root colors due to the accumulation of chlorophylls and anthocyanins compounds that are beneficial for both human health and visual quality. The mechanisms of chlorophyll biosynthesis have been extensively studied in foliar tissues but remain largely unknown in other tissues. In this study, we examined the role of NADPH:protochlorophyllide oxidoreductases (PORs), which are key enzymes in chlorophyll biosynthesis, in radish roots. The transcript level ofRsPORBwas abundantly expressed in green roots and positively correlated with chlorophyll content in radish roots. Sequences of theRsPORBcoding region were identical between white (948) and green (847) radish breeding lines. Additionally, virus-induced gene silencing assay withRsPORBexhibited reduced chlorophyll contents, verifying thatRsPORBis a functional enzyme for chlorophyll biosynthesis. Sequence comparison ofRsPORBpromoters from white and green radishes showed several insertions and deletions (InDels) and single-nucleotide polymorphisms. Promoter activation assays using radish root protoplasts verified that InDels of theRsPORBpromoter contribute to its expression level. These results suggested thatRsPORBis one of the key genes underlying chlorophyll biosynthesis and green coloration in non-foliar tissues, such as roots.