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Abstract In this study, the binding of multimodal chromatographic ligands to the IgG1 F_Cdomain were studied using nuclear magnetic resonance and molecular dynamics simulations. Nuclear magnetic resonance experiments carried out with chromatographic ligands and a perdeuterated¹⁵N‐labeled F_Cdomain indicated that while single‐mode ion exchange ligands interacted very weakly throughout the F_Csurface, multimodal ligands containing negatively charged and aromatic moieties interacted with specific clusters of residues with relatively high affinity, forming distinct binding regions on the F_C. The multimodal ligand‐binding sites on the F_Cwere concentrated in the hinge region and near the interface of the C_H2 and C_H3 domains. Furthermore, the multimodal binding sites were primarily composed of positively charged, polar, and aliphatic residues in these regions, with histidine residues exhibiting some of the strongest binding affinities with the multimodal ligand. Interestingly, comparison of protein surface property data with ligand interaction sites indicated that the patch analysis on F_Ccorroborated molecular‐level binding information obtained from the nuclear magnetic resonance experiments. Finally, molecular dynamics simulation results were shown to be qualitatively consistent with the nuclear magnetic resonance results and to provide further insights into the binding mechanisms. An important contribution to multimodal ligand‐F_Cbinding in these preferred regions was shown to be electrostatic interactions and π–π stacking of surface‐exposed histidines with the ligands. This combined biophysical and simulation approach has provided a deeper molecular‐level understanding of multimodal ligand–F_Cinteractions and sets the stage for future analyses of even more complex biotherapeutics.