The terrestrial plants, Isodon japonicus (Burm. f.) H. Hara and Isodon trichocarpus (Maxim.) Kudô (Labiatae), are native to Japan. Different parts of these plants have been used as a traditional bitter stomachic, under the name Isodon herb (Enmei-so). Ent-kaurane diterpenoids are the major constituents of Isodon herb that contribute to the herb's medicinal properties. However, large variability with respect to the composition of these diterpenoids limits the suitability of Isodon herb as a pharmaceutical ingredient. Thus, an investigation of the factors that affect its chemical composition is required. In this study, the DNA-barcoding method, using internal transcribed spacer sequences of nuclear ribosomal DNA, was applied to cultivated and commercial samples of Isodon herb. Further, each such sample was separated into leaves, stems, and flowers and analyzed for diterpenoid content by HPLC. Moreover, the diterpenoid content in coarsely cut and powdered samples was evaluated. Results confirmed that the source species of these samples was I. japonicus or I. trichocarpus. The three major diterpenoids in Isodon herb were enmein, oridonin, and ponicidin. The diterpenoid content was affected by milling process. Moreover, the diterpenoid content was greatly affected by the ratio between leaves and stems in each sample. Thus, to accurately quantify the diterpenoids in Isodon herb, the use specific conditions such as drying using mild temperature conditions and avoiding milling of the samples might be necessary. This may help in regulating variations in the herb's composition, in turn, providing better quality and a safe herbal product for pharmaceutical use.