Sassafras albidum is an important tree species that occurs across North America. The presence of benzylisoquinoline and aporphine alkaloids has been previously described; however, the spatial distribution of these compounds within S. albidum and other plants of Lauraceae family is still unclear. Mass spectrometry imaging has become an important tool in analysis of plants metabolites, uncovering important contributions about the functional role, biosynthetic pathway, and accumulation of these compounds in the plant. This work aimed to identify further alkaloids present in S. albidum roots, twigs, and leaves by high-performance thin-layer chromatography coupled to desorption electrospray ionization multistage mass spectrometry (HPTLC DESI-MSn) and to map the spatial distribution of these compounds by DESI-MS imaging. A total of 12 alkaloids were indentified in the roots and twigs, and six of them were detected for the first time in S. albidum. A high number of alkaloids was found in S. albidum roots; however, alkaloids were not detected in the leaves. Cross sections of roots and twigs were blotted onto TLC plates assisted by heating and solvent extraction, and these imprints were analyzed by DESI-MS imaging. The profile of alkaloid spatial distribution in DESI-MS images showed different accumulation patterns across and within different plant parts. Most alkaloids displayed higher intensities in the outer-most layer of the roots and twigs. The detailed spatial localization pattern of these alkaloids analyzed by DESI-MS imaging in different plant parts could contribute to a better understanding of the profile of distribution, accumulation, and biosynthesis of benzylisoquinoline and aporphine alkaloids.