The roots of Panax genus with ginseng saponins as bioactive ingredients have been widely used as herbal medicines and food additives. Panax ginseng, Panax quinquefolius, and Panax notoginseng are three major commercial species in Panax genus, with similar morphological appearance but different pharmacological functions. Various methods have been developed and applied for the differentiation of these species. In this work, MALDI-TOF-MS imaging (MSI) was employed for the localization of saponins in root tissues and for the rapid differentiation of the three Panax species for the first time. After a simple sample preparation, MALDI-TOF-MSI analysis of root tissue allowed the detection of 51 saponins. Localization of saponins in the tissue was mapped in ion images, which were obviously related to botanical structure. The localization modes varied with Panax species, providing valuable information for the discrimination of ginseng species. Principal component analysis (PCA) of data collected from areas with abundant saponins based on ion images was applied for the differentiation. Nine characteristic saponin peaks were identified from the PCA analysis. The MALDI-TOF-MSI together with area-specific data analysis provided high potential for the rapid differentiation of Panax herbs.