The distribution of pharmatically important alkaloids gelsemine, koumine, and gelsenicine in tissues is a hot topic attracting research attention. Regretfully, the in planta visual distribution details of these alkaloids are far from clear although several researches reported the alkaloid quantification in by LC-MS/MS. In this study, mass imaging spectrometry (MSI) was employed to visualize the visualization of gelsemine, koumine, and gelsenicine in different organs and tissues of at different growth stages, and the relative quantification of three alkaloids were performed according to the image brightness intensities captured by the desorption electrospray ionization MSI (DESI-MSI).