BackgroundThe genus Celastrus is an important medicinal plant resource. The similarity of morphology and the lack of complete chloroplast genome analysis have significantly impeded the exploration of species identification, molecular evolution and phylogeny of Celastrus. PurposeIn order to resolve the phylogenic controversy of Celastrus species, the chloroplast genome comparative analysis was performed to provide genetic evidence. MethodsIn this study, we collected and sequenced ten chloroplast genomes of Celastrus species from China and downloaded three chloroplast genomes from the databases. The chloroplast genomes were compared and analyzed to explore their characteristics and evolution. Furthermore, the phylogenetic relationships of Celastrus species were inferred based on the whole chloroplast genomes and protein-coding genes. ResultsAll the 13 Celastrus species chloroplast genomes showed a typical quadripartite structure with genome sizes ranging from 155,113 to 157,366 bp. The intron loss of the rps16 gene occurred in all the 13 Celastrus species. The GC content, gene sequence, repeat types and codon bias pattern were highly conserved. Ten highly variation regions were identified, which can be used as potential DNA markers in molecular identification of Celastrus species. Eight genes, including accD, atp4, ndhB, rpoC1, rbcL, rpl2, rpl20 and ycf1, were detected to experience positive selection. Phylogenetic analysis showed that Celastrus was a monophyletic group and Tripterygium was the closest sister-group. Noteworthy, C. gemmatus Loes. and C. orbiculatus Thunb. can be discriminated using the chloroplast genome as a super barcode. The comparative and phylogenetic analysis results proposed that C. tonkinensis Pitard. was the synonym of C. hindsii Benth. ConclusionThe comparative analysis of the Celastrus chloroplast genomes can provide comprehensive genetic evidence for molecular evolution, species identification and phylogenetic relationships.