To reconstruct the phylogenetic relationship of the genus Comastoma and to clarify the species relationship for Comastoma polycladum and its related species. In this study, we sequenced the chloroplast genomes of 12 samples using the Illumina high-throughput sequencing platform, resulting in a large number of high-quality clean reads for subsequent bioinformatics analysis. The findings were as follows: (1) The distinction of chloroplast genome for the species of Comastoma was small, with the 150 kb of length, and we obtained a total of 131 genes, including 81 protein-coding genes (PCGs). The nucleotide polymorphisms in the IR region were lower than in the SC regions, and coding-protein regions were more conserved than non-coding regions. (2) The results of evolutionary analysis indicated that almost all PCGs may be subject to purifying selection. (3) Codon preference analysis revealed that the RSCU values of 35 codons were greater than one, indicating that these codons were used frequently, and codon preference measures revealed that the codon preference was weak. (4) Phylogenetic analysis revealed that the phylogenetic tree constructed using CDS, codon position, and intergenic spacer (IGS) regions datasets had a highly consistent topology, with most branches having a highly support. These results showed that there was no significant difference between the chloroplast genomes of Comastoma polycladum, and its related species, and that it was impossible to cluster them by species on the phylogenetic tree, which provided a scientific basis for future population genetic research on Comastoma.