Siraitia grosvenorii fruit, known as Luo-Han-Guo, has been used as a traditional Chinese medicine for many years, and mogrosides are its primary active ingredients. Unfortunately, Siraitia siamensis, its wild relative, might be misused due to its indistinguishable appearance, not only threatening the reliability of the medication but also partly exacerbating wild resource scarcity. Therefore, high-resolution genetic markers must be developed to discriminate between these species. Here, the complete chloroplast genomes of S. grosvenorii and S. siamensis were assembled and analyzed for the first time; they were 158,757 and 159,190 bp in length, respectively, and possessed conserved quadripartite circular structures. Both contained 134 annotated genes, including 8 rRNA, 37 tRNA and 89 protein-coding genes. Twenty divergences (Pi > 0.03) were found in the intergenic regions. Nine protein-coding genes, accD, atpA, atpE, atpF, clpP, ndhF, psbH, rbcL, and rpoC2, underwent selection within Cucurbitaceae. Phylogenetic relationship analysis indicated that these two species originated from the same ancestor. Finally, four pairs of molecular markers were developed to distinguish the two species. The results of this study will be beneficial for taxonomic research, identification and conservation of Siraitia Merrill wild resources in the future.