explored the downstream e ects of varied partial charge assignment on free energy estimates. Preliminary benchmarks from Open Free Energy Project show that generating partial charges from di erent input conformers leads to variation of up to ±5.3 kcal/mol in calculated relative binding free energies due to variation in partial charges alone. In this study, we more systematically explore this issue, investigating systems where di erences in partial charge generation (such as those caused by input conformer choice, partial charge engine, and hardware) may lead to di erences in calculated absolute hydration free energy (AHFE) values. We demonstrate that supplying di erent input conformers to a partial charge engine can result in atomic partial charge discrepancies of up to 0.681 e, resulting in di erences in calculated AHFE of 6.9 ± 0.1 kcal/mol. We find that even relatively small variations in partial charge assignment can result in notable di erences in calculated AHFE, and thus care should be taken when assigning partial charges to ensure reproducibility and accuracy of any resulting free energy calculations.