Abstract: Gentiana rhodantha is a characteristic medicinal material of Miao Ethnomedicine. It has significant curative effect in the treatment of acute jaundice hepatitis, dysentery, pediatric pneumonia and bronchitis, etc. However, the evolutionary relationship and taxonomic identification of G. rhodantha are controversial. In this study, we sequenced the chloroplast genome of G. rhodantha using the second and third generation sequencing technology. Then, the structural characteristics and suitability evolution characteristics were analyzed. The results showed that the G. rhodantha chloroplast genome was 148 844 bp in length with 37.75% GC content, consisting of a large single copy region (LSC) of 80 076 bp, a small single copy region (SSC) of 17 596 bp and an inverted repeat region (IR) of 25 586 bp. A total of 124 genes were annotated, including 80 protein-coding genes, 36 tRNA genes, and 8 rRNA genes; the chloroplast genome of G. rhodantha has a weak codon preference, and the influencing factors are mainly natural selection. The optimal codons are CUU, UCU, UCA, CCA, and ACU. A total of 169 SSRs were found in MISA, of which the single nucleotide repeats were the most (114, 67.50%), followed by dinucleotide repeats (43, 25.44%). The phylogenetic analysis support that G. rhodantha belong to Sect. Stenogyne which can be clearly distinguished from other groups. Compared with other species, the Ka/Ks value of chloroplast genes of G. rhodantha is basically less than 1 except for psaI, rpl22 and rps11, indicating that they have been subjected to strong purification selection in the long-term evolutionary process. The photosynthesis gene psaI and the expression-related genes rpl22 and rps11 showed differences between groups, which supported the view that Sect. Stenogyne was an independent genus. This study will provide a reference for future researches on chloroplast genetic engineering and molecular breeding of G. rhodantha.