Ginsenosides and lipids are both bioactive ingredients for ginseng. Targeting these two categories of components, this study was designed to develop desorption electrospray ionization-mass spectrometry imaging approaches and spatial metabolomics strategies, achieving the visualization and differentiation among different parts of Panax ginseng root (e.g., rhizome, main root, lateral root, fibrous root, and adventitious root). Potential chemical markers were identified by searching an in-house ginsenoside library and online Lipid Maps database, together with high-resolution MS 2 data analysis. Six ginsenosides and 11 lipids were diagnostic to differentiate five different parts of the P. ginseng root. Additionally, three ginsenosides and 20 lipids were identified as differential markers among the six positions of the main root of P. ginseng. High-abundance malonyl-and oleanolic acid-ginsenosides were observed in the rhizome. These results assist in understanding the accumulation of bioactive molecules all through the root of P. ginseng, which can benefit its quality control and rational use.