Twelve disaccharides containing beta-(1 -> 4)linkages and displaying systematic structural variations in the vicinity of these linkages were selectively labeled with C-13 to facilitate measurements of multiple NMR spin-spin (scalar; J) coupling constants (J(CH) and J(cc) values) across their O-glycosidic linkages. Ensembles of spin-couplings ((2)J(coc), (3)J(cocH), (3)J(cocc)) sensitive to the two linkage torsion angles, phi (phi) and psi (psi), were analyzed by using parametrized equations obtained from density functional theory (DFT) calculations, Fredholm theory, and circular statistics to calculate experiment-based rotamer populations for phi and psi in each disaccharide. With the statistical program MA'AT, torsion angles phi and psi were modeled as a single von Mises distribution, which yielded two parameters, the mean position and the circular standard deviation (CSD) for each angle. The NMR-derived rotamer populations were compared to those obtained from 1 ps aqueous molecular dynamics (MD) simulations and crystallographic database statistical analyses. Conformer populations obtained exclusively from the MA'AT treatment of redundant I-couplings were in very good agreement with those obtained from the MD simulations, providing evidence that conformational populations can be determined by NMR for mobile molecular elements such as O-glycosidic linkages with minimal input from theory. The approach also provides an experimental means to validate the conformational preferences predicted from MD simulations. The conformational behaviors of phi in the 12 disaccharides were very similar, but those of psi varied significantly, allowing a classification of the 12 disaccharides based on preferred linkage conformation in solution.