Understanding the processes that generate novel adaptive phenotypes is central to evolutionary biology. We used comparative analyses to reveal the history of tetrodotoxin (TTX) resistance in TTX-bearing salamanders. Resistance to TTX is a critical component of the ability to use TTX defensively but the origin of the TTX-bearing phenotype is unclear. Skeletal muscle of TTX-bearing salamanders (modern newts, family: Salamandridae) is unaffected by TTX at doses far in excess of those that block action potentials in muscle and nerve of other vertebrates. Skeletal muscle of non TTX-bearing salamandrids is also resistant to TTX but at lower levels. Skeletal muscle TTX-resistance in the Salamandridae results from the expression of TTX-resistant variants of the voltage-gated sodium channel NaV 1.4 (SCN4a). We identified four substitutions in the coding region of salSCN4a that are likely responsible for the TTX-resistance measured in TTX-bearing salamanders and variation at one of these sites likely explains variation in TTX-resistance among other lineages. Our results suggest that exaptation has played a role in the evolution of the TTX-bearing phenotype and provide empirical evidence that complex physiological adaptations can arise through the accumulation of beneficial mutations in the coding region of conserved proteins.