IntroductionIndigoferaL. is the third largest genus in Fabaceae and includes economically important species that are used for indigo dye-producing, medicinal, ornamental, and soil and water conservation. The genus is taxonomically difficult due to the high level of overlap in morphological characters of interspecies, fewer reliability states for classification, and extensive adaptive evolution. Previous characteristic-based taxonomy and nuclear ITS-based phylogenies have contributed to our understanding ofIndigoferataxonomy and evolution. However, the lack of chloroplast genomic resources limits our comprehensive understanding of the phylogenetic relationships and evolutionary processes ofIndigofera.MethodsHere, we newly assembled 18 chloroplast genomes ofIndigofera. We performed a series of analyses of genome structure, nucleotide diversity, phylogenetic analysis, species pairwise Ka/Ks ratios, and positive selection analysis by combining with allied species in Papilionoideae.Results and discussionThe chloroplast genomes ofIndigoferaexhibited highly conserved structures and ranged in size from 157,918 to 160,040 bp, containing 83 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Thirteen highly variable regions were identified, of whichtrnK-rbcL,ndhF-trnL, andycf1 were considered as candidate DNA barcodes for species identification ofIndigofera. Phylogenetic analysis using maximum likelihood (ML) and Bayesian inference (BI) methods based on complete chloroplast genome and protein-coding genes (PCGs) generated a well-resolved phylogeny ofIndigoferaand allied species.Indigoferamonophyly was strongly supported, and four monophyletic lineages (i.e., the Pantropical, East Asian, Tethyan, and Palaeotropical clades) were resolved within the genus. The species pairwise Ka/Ks ratios showed values lower than 1, and 13 genes with significant posterior probabilities for codon sites were identified in the positive selection analysis using the branch-site model, eight of which were associated with photosynthesis. Positive selection ofaccDsuggested thatIndigoferaspecies have experienced adaptive evolution to selection pressures imposed by their herbivores and pathogens. Our study provided insight into the structural variation of chloroplast genomes, phylogenetic relationships, and adaptive evolution inIndigofera. These results will facilitate future studies on species identification, interspecific and intraspecific delimitation, adaptive evolution, and the phylogenetic relationships of the genusIndigofera.