Abstract

Background/Objectives:TheFagopyrum dibotryscomplex is a specialized high-altitude lineage in southwestern China with medicinal and breeding potential, but species delimitation remains unresolved.Methods:We sequenced 26 complete chloroplast genomes from the Hengduan Mountains to the Yunnan–Guizhou Plateau, analyzing genomic structures, variation patterns, and phylogenetic relationships.Results:All genomes exhibited typical quadripartite structures (152,213–160,302 bp), containing 133 genes (88 protein-coding, 8 rRNA, and 37 tRNA) with GC content of 37.9%. Collinearity analysis revealed highly conserved structures without structural rearrangements. Variations were concentrated in the large single-copy(LSC)/small single-copy(SSC) non-coding regions, with hotspots atycf4–cemAandndhF–rpl32. Codon usage showed an A/U bias, with leucine being most abundant and cysteine the least. Simple sequence repeats (SSRs) were predominantly mononucleotide repeats enriched in the LSC, while long repeats were mainly palindromic/forward. Maximum likelihood and Bayesian phylogenies consistently resolved three clades: Tibetan high-altitude specialists, limestone specialists, and a widespread Hengduan–Yunnan–Guizhou clade, with geographic clustering indicating isolation as the primary differentiation driver.Conclusions:This study refines the phylogenetic resolution of theF. dibotryscomplex and identifies informative chloroplast markers, providing a genomic foundation for reliable species delimitation, evolutionary inference, and conservation management of this medicinal lineage.