Spatial organization of polymer chains upon ionic or covalent cross-linking to form a hydrogel is examined on the molecular scale using a fluorescent resonance energy transfer (FRET) technique. Minimal changes in the conformation in the polymer chains but significant changes of the intermolecular association of polymer chains are found when polymer chains are cross-linked with a different number of cross-links. This study provides a previously undescribed technique to analyze hydrogel structure in a noninvasive manner.