Zhemaidong (ZMD) and Chuanmaidong (CMD) are the two genuine cultivation areas of Ophiopogonis Radix which has been widely used as a traditional Chinese medicine in China for treating cardiovascular and pulmonary diseases. Differences between ZMD and CMD in chemical constituents and pharmacological effects have been reported, however, the details remain largely unknown. The aim of this study was to comprehensively characterize the chemical composition of Ophiopogonis Radix from the two producing areas and compare their immunomodulatory activities. An approach of HPLC–MS coupled with multivariate statistical analysis was established to reveal the characteristic constituents of ZMD and CMD. Furthermore, the effects of ZMD and CMD on the macrophage phagocytosis and gastrointestinal peristalsis were also determined in zebrafish models for assessing the immunomodulatory activities of these two strains. The results revealed that the chemical constituents of ZMD and CMD were much different from each other, and 19 constituents could be served as chemical markers to distinguish these two strains. Moreover, ZMD showed higher promoting rates in macrophage phagocytosis and gastrointestinal motility than those of CMD, suggesting ZMD might possess better immunomodulatory activities. Taken together, the results generated from this study indicated that the herbs from different producing areas should be evaluated and considered in preparing TCM prescriptions.