Layer-stacking structures are very common in two-dimensional covalent organic frameworks (2D COFs). While their structures are normally determined under solvent-free conditions, the structures of solvated 2D COFs are largely unexplored. We report herein the in situ determination of solvated 2D COF structures, which exhibit an obvious difference as compared to that of the same COF under dried state. Powder X-ray diffraction (PXRD) data analyses, computational modeling, and Pawley refinement indicate that the solvated 2D COFs experience considerable interlayer shifting, resulting in new structures similar to the staggered AB stacking, namely, quasi-AB-stacking structures, instead of the AA-stacking structures that are usually observed in the dried COFs. We attribute this interlayer shifting to the interactions between COFs and solvent molecules, which may weaken the attraction strength between adjacent COF layers. Density functional theory (DFT) calculations confirm that the quasi-AB stacking is energetically preferred over the AA stacking in solvated COFs. All four highly crystalline 2D COFs examined in the present study exhibit considerable interlayer shifting upon solvation, implying the universality of the solvent-induced interlayer stacking rearrangement in 2D COFs. These findings prompt re-examination of the 2D COF structures in solvated state and suggest new opportunities for the applications of COF materials under wet conditions.