Brassica juncea is an allopolyploid originating from the interspecific hybridization between Brassica rapa and Brassica nigra, which is of multiple usage as a vegetable, oilseed and condiment worldwide. Both vernalization and non-vernalization under long-day photoperiod can promote floral transition in B. juncea suggesting merged flowering pathways of its ancestors and better environmental adaptability. We identified genome-wide flowering regulatory genes in B. juncea, which include 84 and 79 genes from A and B sub-genomes, respectively. Ka/Ks analysis revealed a purification effect on both photoperiod and vernalization flowering regulation pathways during evolution. Expression profile of those genes during long-day and vernalization treatments suggested BjuACO4, BjuAFT1, BjuBFT4, BjuASOC1 and BjuASOC4 may be the major functional copies of B. juncea flowering regulation. Further functional studies about BjuCOs showed three days delayed flowering time in BjuACO4 or BjuBCO3 silenced plants. Increased transcription of all BjuFLCs in BjuACO4 or BjuBCO3 silenced plants suggested interactions between photoperiod and vernalization pathways governing flowering time. Our findings provided flowering regulating networks in allopolyploid B. juncea.