The traditional visual identification of Chinese herbs is not objective. Molecular biological techniques can be used to accurately identify the origin of each herbal species. This can enable the further purification and control of important herbal species. Molecular techniques involving polymerase chain reaction and sequencing were used to provide a relatively simple and objective means of identifying burdock at the species level. This study proved that the length of the ITS1–5.8S rRNA-ITS2 sequence was 358 base-pairs (bp) for six types of Arctium lappa Linn (the following breeds: Pingtung Gueilai, General Snow, Japanese, Fengshan, Wholesaler, and Tainan). Automatic sequencing analysis found that ITS DNA sequences for Pingtung Gueilai and Japanese breeds were the same, and the General Snow breed differed from them at its 277 bp. This study used DNA sequencing to analyze the high specificity regions of A. lappa Linn ITS, originated in different parts of Taiwan, and discovered the breed identification single-nucleotide polymorphism at the 277 bp position for local differentiation.