The function of structured RNA molecules depends on forming the correct 3D structure, so the most significant constraints on their sequences are structural. Structure-disrupting substitutions are selected against during evolution while structurally neutral substitutions can accumulate as populations evolve. The relevant interactions include basepairs, base-stacking, and base-phosphate interactions, all of which can be disrupted by certain substitutions. Further constraints are imposed by interactions with other molecules. The general question we address is how to determine which substitutions are structure-neutral in RNA molecules, at the level of individual bases and base-pairs and at the level of 3D motifs and molecular architectures. The availability of two or more 3D structures of large RNA molecules such as the 16S and 23S rRNAs presents opportunities for exploring this question empirically, once the two structures are appropriately aligned. Detailed examination and comparison of nucleotide-nucleotide interaction geometries provides another avenue for addressing the same question. Finally, some RNA motifs occur multiple times in single structures and in non-homologous positions in other molecules, giving another way to study the neutrality of base substitutions. These three approaches will be described and their results compared. Along the way, we will briefly describe FR3D (“Find RNA 3D”), a set of Matlab programs we have developed to annotate RNA structures and to carry out searches for recurrent RNA motifs.