Herein, we reported the designed synthesis of three isostructural three-dimensional covalent organic frameworks (3D COFs) with -H, -Me,or-Fsubstituents,which have similar crystallinity and topology.T heir crystal structures were determined by continuous rotation electron diffraction (cRED), and all three 3D COFs were found to adopt af ivefold interpenetrated pts topology.M ore importantly,t he resolution of these cRED datasets reached up to 0.9-1.0 ,e nabling the localization of all non-hydrogen atomic positions in aC OF framework directly by 3D ED techniques for the first time.In addition, the precise control of the pore environments through the use of different functional groups led to different selectivities for CO 2 over N 2 .W eh ave thus confirmed that polycrystalline COFs can be definitely studied to the atomic level as other materials,a nd this study should also inspire the design and synthesis of 3D COFs with tailored pore environments for interesting applications.