Horticultural plants synthesize diverse flavonoids (e.g. anthocyanins, proanthocyanidins, and flavonols) that shape key agronomic traits, including flower pigmentation, fruit flavor, stress resilience, and nutritional value. MYB transcription factors (TFs), particularly R2R3-MYBs, serve as core regulators of flavonoid biosynthesis; their functional divergence and distinct regulatory mechanisms drive the interspecific diversity of flavonoid-related traits. This review systematically summarizes the evolutionary and functional divergence of flavonoid-regulatory R2R3-MYBs across monocot (including Poaceae and non-Poaceae) and eudicot horticultural plants. Phylogenetic analysis, integrating functionally validated R2R3-MYB sequences and Arabidopsis references, classifies the R2R3-MYB family into 37 subgroups. Flavonoid-regulatory members primarily cluster within key dedicated subgroups, which are first categorized into six functional groups and subsequently subdivided into 11 distinct subclades based on their monophyletic distribution in angiosperms across 44 horticultural species. Distinct lineage-specific evolutionary patterns emerge: some monocots (notably Poaceae) putatively lack Subgroup 6 (SG6) and rely on SG5 for anthocyanin biosynthesis, while eudicots expand SG6 via tandem duplications. Rosaceae exhibit adaptive expansion of SG5 associated with fruit trait evolution. We further discuss cross-species divergence within conserved flavonoid-regulatory subgroups, including variations in spatiotemporal expression patterns and dependency on the MYB–bHLH–WD40 (MBW) complex. Case studies of representative species (Arabidopsis, rice, strawberry, and freesia) demonstrate that paralogs derived from gene duplication achieve precise regulation of distinct flavonoid branch pathways through subfunctionalization or neofunctionalization. Ecological pressures, domestication, and structural determinants collectively drive MYB divergence in copy number and function. Finally, we outline the applications of these findings in crop improvement and propose future research directions.

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