Citrus reticulata cv. Chachiensis (CRC) is an important medicinal plant, with its dried mature peels, known as "Guangchenpi," used in traditional Chinese medicine for treating cough, indigestion, and lung diseases. However, the biosynthesis of polymethoxylated flavonoids (PMFs), crucial natural products in CRC, remains unclear. This study reports a chromosome-scale genome assembly of CRC, with a size of 314.96 Mb and a contig N50 of 16.22 Mb. Using multi-omics resources, the researchers identified a putative caffeic acid O-methyltransferase (CcOMT1) that can transfer a methyl group to the 3-hydroxyl of natsudaidain to form 3,5,6,7,8,3',4'-heptamethoxyflavone (HPMF). Through transient overexpression and virus-induced gene silencing experiments, they propose that CcOMT1 is a candidate enzyme in HPMF biosynthesis. Additionally, a potential gene regulatory network associated with PMF biosynthesis was identified. This study provides insights into PMF biosynthesis and may assist future research on mining genes for the biosynthesis of plant-based medicines.Citrus reticulata cv. Chachiensis (CRC) is an important medicinal plant, with its dried mature peels, known as "Guangchenpi," used in traditional Chinese medicine for treating cough, indigestion, and lung diseases. However, the biosynthesis of polymethoxylated flavonoids (PMFs), crucial natural products in CRC, remains unclear. This study reports a chromosome-scale genome assembly of CRC, with a size of 314.96 Mb and a contig N50 of 16.22 Mb. Using multi-omics resources, the researchers identified a putative caffeic acid O-methyltransferase (CcOMT1) that can transfer a methyl group to the 3-hydroxyl of natsudaidain to form 3,5,6,7,8,3',4'-heptamethoxyflavone (HPMF). Through transient overexpression and virus-induced gene silencing experiments, they propose that CcOMT1 is a candidate enzyme in HPMF biosynthesis. Additionally, a potential gene regulatory network associated with PMF biosynthesis was identified. This study provides insights into PMF biosynthesis and may assist future research on mining genes for the biosynthesis of plant-based medicines.