Ophiopogon japonicus, also called “Maidong” (MD) in Chinese, is a well known Chinese medicine which possesses important pharmacological effects including cardiovascular protection, anti-tumor and anti-diabetes, etc [1]. In the market, Ophiopogon japonicus could come from two major production places Zhejiang (ZMD) and Sichuan (CMD), and studies have proved that the active components in the two production places are different [2,3]. Furthermore, its close species Liriope spicata (SMD) may present as substitution or adulterants in the market, making quality control of Ophiopogon japonicus even more difficult. In this study, headspace-gas chromatography-mass spectrometry (HS-GC-MS) method was used to study the volatile organic compounds (VOCs) in ZMD, CMD and SMD; multivariate statistical analyses including PCA, OPLS-DA and PLS were used to compare the differences among the VOCs profiles and significant chemical markers were screened for discrimination of the three samples. In general, there were significant differences between the VOCs profiles of 49 samples including ZMD, CMD and SMD, and a total of 1780 variables were successfully applied to discriminate the three kinds of samples; furthermore, several chemical markers among the variables were screened using OPLS-DA and PLS, and the markers were proved to be able to discriminate MD from SMD, and MD from different regions or of different ages. In conclusion, the developed HS-GC-MS coupled with metabolomics method could be used for discrimination of MD from different producing regions and its analogue, as well as identification of their differentiation markers. When compared with traditional GC, the developed method in this study provided a rapid and easily applicable option for daily routine quality analysis of MD, and might be furtherly applied for quality study of other Chinese medicines.