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• Transcriptomic analysis from five different tissues reveal candidate unigenes involved in flavonoid metabolism. • Flavonoid profiles show the accumulation of the active ingredients in Chrysanthemum indicum. • One flavone synthases (FNSⅡ) was identified which catalyzes the conversion of flavanones to flavones. • A pharmacological component-linarin biosynthesis pathway is proposed. • Lay a foundation of potentials for molecular breeding and metabolic engineering in C. indicum. Chrysanthemum indicum L. is a type of herb that is widely used in China, Korea, and Japan. It has been used as an ingredient in traditional medicines, tea, and functional food because of its various anti-inflammatory and anti-oxidant bioactivities. Such bioactivities have been associated with flavonoids such as apigenin, luteolin and linarin in C. indicum. However, the biosynthesis pathway has not been investigated. In this study, using transcriptomic analysis and targeted metabolic profiling from five different tissues, we characterize the levels of flavonoids and mine the corresponding genes involved in flavonoid biosynthesis. Transcriptomic analysis revealed that 103 unigenes are involved in flavonoid-related biosynthesis pathways. Flavone synthase (FNS) is the key enzyme responsible for flavone synthesis and provides precursors for acacetin and linarin biosynthesis. One putative FNS Ⅱ gene, with the highest Reads Per Kilobase per Million mapped reads (RPKM) in flower and flower bud was cloned. Quantitative real-time polymerase chain reaction (RT-qPCR) revealed that CiFNSⅡ exhibited a similar expression pattern to that in the transcriptome in terms of RPKM. In addition, a targeted metabolic profiling of three flavanones (naringenin, eriodictyol, and liquiritigenin), three flavones (apigenin, luteolin, and 7,4′-dihydroxyflavone), and two flavone derivatives (linarin and acacetin) was performed to characterize the distribution of these flavonoids in different tissues of C. indicum. The recombinant FNSⅡ protein expressed in yeast was able to catalyze the conversion of three flavanones into the respective flavones. Based on the transcriptome analysis, metabolic profiling, and activity assays, a linarin biosynthesis pathway is proposed. Our study provides insight into the potential application of molecular breeding and metabolic engineering for improving the quality of cultivated C. indicum. [ABSTRACT FROM AUTHOR]