Within some species, squirrels respond to variable selection from venomous snake predators by showing population-level variation in resistance, while between species, some rattlesnakes possess venom that is more effective at overcoming venom resistance in different species of squirrels. A functional evaluation of resistance variation to venom within and between species of squirrels and snakes can link resistance variation to its evolutionary causes across these different evolutionary scales. To do this, we compared the effectiveness of squirrel sera in inhibiting rattlesnake (Crotalus spp.) venom metalloproteinase activity between populations and between species to test for a response to local variation in selection from a single rattlesnake predator and for specialization of two resistant squirrel species to each of their distinct sympatric snake predators. We found that Timber Rattlesnake (Crotalus horridus) venom inhibition by Eastern gray squirrels (Sciurus carolinensis) is higher at a site where the rattlesnakes are present, which suggests selection may maintain venom resistance in populations separated by short distances. Next, we performed a reciprocal cross of venoms and sera from two rattlesnake and two squirrel species. This showed that squirrel resistance is lower when tested against venom from allopatric compared to sympatric rattlesnake species, demonstrating that squirrel inhibitors are specialized to sympatric venom and suggesting a tradeoff in terms of specialization to the venom of a specific species of rattlesnake predator. This pattern can be explained if inhibitors must recognize venom proteins and resistance evolution tracks venom evolution.