Proteoglycans (PGs) are covalent conjugates between protein and carbohydrate (glycosaminoglycans). Certain classes of glycosaminoglycans such as chondroitin sulfate/dermatan sulfate and heparan sulfate utilize a specific tetrasaccharide linker for attachment to the protein component: GlcAβ1‐3Galβ1‐3Galβ1‐4Xylβ1‐O‐Ser. Toward understanding the conformational preferences of this linker, the present work used all‐atom explicit‐solvent molecular dynamics (MD) simulations combined with Adaptive Biasing Force (ABF) sampling to determine high‐resolution, high‐precision conformational free energy maps ΔG(φ, ψ) for each glycosidic linkage between constituent disaccharides, including the variant where GlcA is substituted with IdoA. These linkages are characterized by single, predominant (> 97% occupancy), and broad (45° × 60° for ΔG(φ, ψ) < 1 kcal/mol) free‐energy minima, while the Xyl‐Ser linkage has two such minima similar in free‐energy, and additional flexibility from the Ser sidechain dihedral. Conformational analysis of microsecond‐scale standard MD on the complete tetrasaccharide‐O‐Ser conjugate is consistent with ABF data, suggesting (φ, ψ) probabilities are independent of the linker context, and that the tetrasaccharide acts as a relatively rigid unit whereas significant conformational heterogeneity exists with respect to rotation about bonds connecting Xyl to Ser. © 2017 Wiley Periodicals, Inc.