Ophiopogonis Radix (Maidong in Chinese), the tuberous root of Ophiopogon japonicus, is a widely used traditional Chinese herb. The major components of Maidong include steroidal saponins, homoisoflavonoids and polysaccharides, which exhibited various pharmacological activities, such as cardiovascular protection, anti-inflammation, anticancer, immunomodulation, and anti-diabetes. In China, Maidong is mainly produced in Sichuan and Zhejiang provinces, where it is called Chuanmaidong or Zhemaidong respectively. Traditionally, the quality of Zhemaidong is considered the best, but the production of Chuanmaidong is the biggest at present. In addition, Liriopes Radix (Shanmaidong in Chinese) is usually used as a substitute for Maidong in traditional Chinese medicine. Therefore, quality control is crucial to ensure the safety and efficacy of Maidong in clinical applications. In this study, TLC fingerprint and HPLC method for quantitation of characteristic components in Maidong were developed. The results showed the chemical characteristics of Chuanmaidong, Zhemaidong and Shanmaidong are significantly different. Methylophiopogonanone A and B are only detected in Maidong, but not in Shanmaidong, which could be used for discrimination of these two herbs. Zhemaidong contains higher contents of methylophiopogonanone A and B than Chuanmaidong; While the content of ophiopogonin D, the marker for quality control of Maidong in Hong Kong Chinese Materia Medica Standards, is higher in Chuanmaidong, but very low or not detected in Zhemaidong. This work is helpful for the quality control of Maidong and provides information as a basis of further development and utilization of this herbal medicine.