Pinellia Tuber is defined as Pinellia ternata Breitenbach (Family Araceae), according to the Korean Pharmacopoeia (KP). Recent studies have reported the mass production of Pinellia Tuber using tissue culture technology. However, somatic variations during tissue culture have been reported to produce tuber forms resembling those of P. pedatisecta. Additionally, the potential for closely related species, such as P. tripartita and Typhonium flagelliforme, to be distributed as Pinellia Tuber should be monitored carefully for species identification. In this study, we identified indel (insertion/deletion) regions through a comparative analysis of chloroplast genomes and developed specific markers to distinguish P. ternata (182 bp) from P. pedatisecta (352 bp) and P. tripartita (388 bp). Using the developed marker (CP_2F/2R), we established a genetic analysis method that enables the effective and rapid identification of Pinellia Tuber and its potential substitutes with a single PCR reaction. This method can also detect the presence of P. pedatisecta contamination in mixed samples. The proposed approach is expected to contribute to the quality control of Pinellia Tuber, a frequently prescribed herbal medicine, and to prevent misidentification or adulteration.