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<i>Rosa roxburghii</i> Tratt, the most popular fruit that blooms in the southwest of China, has high antioxidant properties and is rich in different flavonoids. However, the regulatory network and critical genes that regulate the flavonoid biosynthesis of <i>R. roxburghii</i> are still unknown. In this study, HPLC analysis revealed that total flavonoids, anthocyanins, and catechin were enriched in mature fruits, flowers, and leaves, respectively. Differentially expressed genes (DEGs) between five organs of <i>R. roxburghii</i> involved in flavonoid metabolism were obtained by transcriptome sequencing. A total of 1130 DEGs were identified, including 166 flavonoid pathway biosynthesis genes, 622 transcription factors (TFs), 301 transporters, and 221 cytochrome P450 proteins. A weighted gene co-expression network analysis (WGCNA) of the DEGs was conducted to construct co-expression networks. Regarding enzymes in the biosynthesis of flavonoids, cytochrome P450 <i>CYP749A22</i> and <i>CYP72A219</i> were highlighted in the regulation of total flavonoids of mature fruits. Anthocyanin 3-O-glucosyltransferase and F3′H were the top two critical enzymes for anthocyanin accumulation in flowers. By contrast, caffeic acid 3-O-methyltransferase, 4-coumarate-CoA ligase, and shikimate O-hydroxycinnamoyltransferase were essential for catechin accumulation in leaves. Additionally, we analyzed the eigengene network of the “black” module, which had high correlations with total flavonoids (r = 0.9, <i>p</i> = 5 × 10⁻⁶). There were 26 eigengenes in the “black” module, consisting of 6 flavonoid biosynthesis, 14 TFs, and 6 transporters. Among them, the transcription factors <i>RrWRKY45</i> (<i>DN142829_c1_g5</i>), <i>RrTCP20</i> (<i>DN146443_c1_g1</i>), and <i>RrERF118</i> (<i>DN141507_c3_g2</i>) were screened as the hub genes, which significantly correlated with total flavonoids in <i>R. roxburghii</i>. The present biochemical and transcriptomic data provide insights into functional genomics for breeding <i>R. roxburghii</i> with flavonoid accumulation.