Abstract: The whole chloroplast genome of the medicinal plant Paeonia mairei H. Lév. was sequenced using the Illumina HiSeq X Ten platform and then assembled, annotated, and characterized by bioinformatic methods in this study. The complete chloroplast genome of P. mairei is 152 731 bp in length with the typical quadripartite structure, which consists of a large single copy-region (LSC, 84 402 bp), a small single copy-region (SSC, 16 969 bp), and a pair of inverted repeat regions (IRa and IRb, 25 680 bp), with an overall GC content of 38.4%. A total of 136 predicted genes, including 90 protein-coding genes, 38 tRNA genes and eight rRNA genes were identified. Among these, seven protein-coding genes, seven tRNA genes and four rRNA genes were found duplicated in the IR regions. In addition, 28 dispersed repeats, 10 tandem repeats, and 64 simple sequence repeats were detected within the whole chloroplast genome of P. mairei. Comparative analyses between 12 Peaonia species showed that the chloroplast genomes are highly conserved in length, gene content, gene order, and GC content. Meanwhile, the noncoding sequences (intergenic regions and introns) show a higher variation than the protein coding sequences, and sequences from the LSC region and SSC region are more variable than those from the IR regions. P. mairei was inferred forming in a distinct clade with P. lactiflora, P. obovate, and P. anomala subsp. veitchii with a 100% bootstrap value and is phylogenetically closest to P. lactiflora. These results may provide a basis for further genetic studies and the development and utilization of medicinal P. mairei.