Rubia cordifolia L., a plant in the Rubiaceae family. In this study, the whole chloroplast genome was sequenced by the high throughput sequencing technology of Illumination, and bioinformatics tools such as MAGA11 were used for genome analysis and phylogenetic analysis, so as to provide data reference for identification, genetic diversity analysis and resource exploitation and utilization of Rubia cordifolia and its related medicinal plants. The results showed that: (1) The chloroplast genome of Rubia cordifolia L. showed a typical quadripartite ring structure with a total GC content of 37.2%, a length of 153 959 bp, including a large single copy (LSC) region of 83 844 bp, a small single copy (SSC) region of 17 083 bp and two reverse repeat (IR) regions of 26 516 bp. A total of 124 genes were annotated, including 79 protein-coding genes, 37 tRNAs and 8 rRNAs. (2) A total of 169 SSRs were detected, mainly composed of A and T, including 129 mononucleotides, 18 dinucleotides, 11 trinucleotides, 9 tetranucleotides and 2 pentanucleotides, but no hexonucleotides were detected by SSR. Boundary analysis showed that the LSC region of Rubia cordifolia chloroplast genome had the largest difference and the highest degree of variation, while the IRa region had the smallest difference and was the most conservative. (3) The maximum likelihood method (ML) was used to construct phylogenetic tree, and the tree shows that Rubia cordifolia was grouped with the same species Rubia horrida with 100% support. The Rubioideae, Ixoroideae, and Cinchonoideae form the sister group, demonstrating that Rubiaceae has developed conserved during evolution. The results can provide data reference for identification, genetic diversity analysis and resource exploitation of Rubia cordifolia and its related species.