To systematically determine their phylogenetic relationships and develop molecular markers for species discrimination of Salvia bowleyana, S. splendens, and S. officinalis, we sequenced their chloroplast genomes using the Illumina Hiseq 2500 platform. The chloroplast genomes length of S. bowleyana, S. splendens, and S. officinalis were 151,387 bp, 150,604 bp, and 151,163 bp, respectively. The six genes ndhB, rpl2, rpl23, rps7, rps12, and ycf2 were present in the IR regions. The chloroplast genomes of S. bowleyana, S. splendens, and S. officinalis contain 29 tandem repeats; 35, 29, 24 simple-sequence repeats, and 47, 49, 40 interspersed repeats, respectively. The three specific intergenic sequences (IGS) of rps16-trnQ-UUG, trnL-UAA-trnF-GAA, and trnM-CAU-atpE were found to discriminate the 23 Salvia species. A total of 91 intergenic spacer sequences were identified through genetic distance analysis. The two specific IGS regions (trnG-GCC-trnM-CAU and ycf3-trnS-GGA) have the highest K2p value identified in the three studied Salvia species. Furthermore, the phylogenetic tree showed that the 23 Salvia species formed a monophyletic group. Two pairs of genus-specific DNA barcode primers were found. The results will provide a solid foundation to understand the phylogenetic classification of the three Salvia species. Moreover, the specific intergenic regions can provide the probability to discriminate the Salvia species between the phenotype and the distinction of gene fragments.

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