In this study, we explored the relationship between the retention factors and structural flexibilities of peptide diastereomers in reversed-phase chromatography (RPC) based on thermodynamic interpretations. The RPC retention order of antimicrobial peptides, IL-K7F89 (H-ILPWKWKFFPWRR-NH2), and its four diastereomers were well correlated with the order of their conformation energies in elution solvent. In particular, when the composition of the sample loading solvent was altered, the retention order changed accordingly. The thermodynamic analysis revealed that the peptide adsorption was driven by adsorption enthalpy, but the retention order was dominated by adsorption entropy. To further understand the relationships between the retention factor and conformation energy, the intramolecular van der Waals energy of peptides and the ordered water molecules associated with peptides were analyzed by all-atom molecular dynamics (MD) simulation. The results showed that the flexible peptide with larger conformation energy had weaker intramolecular hydrophobic interaction and associated with more ordered water molecules. For this peptide diastereomer set, the elution difference is derived by the difference in adsorption entropy gain from repelling the ordered water molecules around the peptide. Consequently, we suggested that the separation of peptide diastereomers could be achieved by RPC, and the elution order could be predicted by their structural flexibilities.