Voltage-gated sodium channels (NaVs) play an important role in the electric signaling of cells by allowing the movement of ions across cellular membranes in response to changes in membrane potential. The nine human NaV subtypes perform distinct physiological functions and are of substantial therapeutic interest due to their involvement in several physical conditions, such as neurological and cardiac disorders, as well as nociception. Many clinically relevant compounds target NaVs, including the pufferfish toxin tetrodotoxin (TTX), a highly potent and selective channel blocker with potential applications in neuropathic pain management. Despite their amino acid sequence being highly conserved, certain NaV subtypes, such as NaV1.6 and NaV1.7, are highly sensitive to TTX, with IC50 values in the nanomolar range, while others, such as Nav1.5 and NaV1.8 are resistant to TTX. Understanding the molecular mechanism for NaV-TTX selectivity is important for development of TTX-based pain therapies. Using recent cryo- electron microscopy structures of TTX-bound NaV channels, we use molecular dynamics (MD) simulations and free energy perturbation (FEP) to investigate the key molecular interactions which dictate TTX selectivity, comparing to experimental KD values. These insights establish a molecular basis for further developing TTX-derived compounds for pain therapeutics.