Lotus predominantly accumulates benzylisoquinoline alkaloids (BIAs), but their biosynthesis and regulation remain unclear. Here, we investigated structural and regulatory genes involved in BIA accumulation in lotus. Two clustered CYP80 genes were identified to be responsible for the biosynthesis of bis-BIAs and aporphine-type BIAs, respectively, and their tissue-specific expression causes divergence in alkaloid component between leaf and embryo. In contrast with the common (S)-reticuline precursor for most BIAs, aporphine alkaloids in lotus leaf may result from the (S)-N-methylcoclaurine precursor. Structural diversity of BIA alkaloids in the leaf is attributed to enzymatic modifications, including intramolecular C-C phenol coupling on ring A and methylation and demethylation at certain positions. Additionally, most BIA biosynthetic pathway genes show higher levels of expression in the leaf of high-BIA cultivar compared with low-BIA cultivar, suggesting transcriptional regulation of BIA accumulation in lotus. Five transcription factors, including three MYBs, one ethylene-responsive factor, and one basic helix-loop-helix (bHLH), were identified to be candidate regulators of BIA biosynthesis in lotus. Our study reveals a BIA biosynthetic pathway and its transcriptional regulation in lotus, which will enable a deeper understanding of BIA biosynthesis in plants.