A multi-omics approach was used to study the biosynthesis of polymethoxylated flavonoids (PMFs) in Citrus reticulata cv. Chachiensis (CRC), a medicinal plant used in traditional Chinese medicine. A chromosome-scale genome of CRC was assembled, revealing a genome size of 314.96 Mb with a contig N50 of 16.22 Mb. Using multi-omics data, a putative caffeic acid O-methyltransferase (CcOMT1) was identified as a candidate enzyme in the biosynthesis of HPMF, a key PMF with potential anticancer and antioxidant properties. A gene regulatory network associated with PMF biosynthesis was also identified. The study provides insights into PMF biosynthesis and may assist future research on the genetic basis of plant-based medicines. CRC is a variety of citrus fruit with high medicinal value, particularly for its dried peels, "Guangchenpi," used to treat cough, digestive issues, and lung diseases. PMFs, which are key components of "Guangchenpi," have various bioactivities, including anticancer, neuroprotective, and anti-inflammatory effects. However, the biosynthesis of PMFs in citrus fruits remains poorly understood. This study identified 47 high-confidence OMT genes, with CcOMT1 being a key enzyme in the biosynthesis of HPMF. In vitro and in vivo experiments confirmed that CcOMT1 can catalyze the methylation of the 3-hydroxyl group of natsudaidain to produce HPMF. The study also identified a potential gene regulatory network for PMF biosynthesis, involving transcription factors such as AP2/ERF, bZIP, and WRKY, which may regulate flavonoid biosynthesis and accumulation. The study also revealed that the expression levels of key regulatory genes, such as AP2/ERF-ERF, LIM, and HB-HD-ZIP, are higher in the core region of CRC production, which may contribute to the higher quality of "Guangchenpi" in this region. The proposed biosynthetic pathway of PMFs in CRC starts with naringenin, which is modified by various enzymes to produce different PMFs, including HPMF. The study provides a comprehensive understanding of the biosynthesis of PMFs in CRC, which may aid in the improvement of "Guangchenpi" quality and the development of new medicinal applications. The multi-omics data generated in this study will also contribute to future research on the genetic basis of plant-based medicines.A multi-omics approach was used to study the biosynthesis of polymethoxylated flavonoids (PMFs) in Citrus reticulata cv. Chachiensis (CRC), a medicinal plant used in traditional Chinese medicine. A chromosome-scale genome of CRC was assembled, revealing a genome size of 314.96 Mb with a contig N50 of 16.22 Mb. Using multi-omics data, a putative caffeic acid O-methyltransferase (CcOMT1) was identified as a candidate enzyme in the biosynthesis of HPMF, a key PMF with potential anticancer and antioxidant properties. A gene regulatory network associated with PMF biosynthesis was also identified. The study provides insights into PMF biosynthesis and may assist future research on the genetic basis of plant-based medicines. CRC is a variety of citrus fruit with high medicinal value, particularly for its dried peels, "Guangchenpi," used to treat cough, digestive issues, and lung diseases. PMFs, which are key components of "Guangchenpi," have various bioactivities, including anticancer, neuroprotective, and anti-inflammatory effects. However, the biosynthesis of PMFs in citrus fruits remains poorly understood. This study identified 47 high-confidence OMT genes, with CcOMT1 being a key enzyme in the biosynthesis of HPMF. In vitro and in vivo experiments confirmed that CcOMT1 can catalyze the methylation of the 3-hydroxyl group of natsudaidain to produce HPMF. The study also identified a potential gene regulatory network for PMF biosynthesis, involving transcription factors such as AP2/ERF, bZIP, and WRKY, which may regulate flavonoid biosynthesis and accumulation. The study also revealed that the expression levels of key regulatory genes, such as AP2/ERF-ERF, LIM, and HB-HD-ZIP, are higher in the core region of CRC production, which may contribute to the higher quality of "Guangchenpi" in this region. The proposed biosynthetic pathway of PMFs in CRC starts with naringenin, which is modified by various enzymes to produce different PMFs, including HPMF. The study provides a comprehensive understanding of the biosynthesis of PMFs in CRC, which may aid in the improvement of "Guangchenpi" quality and the development of new medicinal applications. The multi-omics data generated in this study will also contribute to future research on the genetic basis of plant-based medicines.