Traditional micro- and macrodissection techniques enable the extraction of localized regions in thin tissue sections for molecular analysis. Despite the growing use of three-dimensional (3D) microscopy, analogous methods for volumetric microdissection are lacking. Here we have developed a 3D microdissection method based on computer numerical controlled milling integrated with open-top light-sheet microscopy. We demonstrate the ability to study tumor evolution along convoluted 3D branching architectures, which is inaccessible to two-dimensional methods.