In mammals, expression of certain melanocortin receptor ligands is correlated with both dark pigmentation and increased stress resistance and higher levels of aggression. Though many studies of captive and laboratory animals have explored this pleiotropic interaction, relatively few studies of animal behavior have occurred in free-living wild animals. This playback study focused on the antipredator behavior differences between melanistic and gray morphs of eastern gray squirrels (Sciurus carolinensis) in Hiram, Ohio. Vigilance, tail flagging, freezing, and escape behaviors were recorded in response to digital playback of an American robin call, a chickadee call, a car alarm, a buzzer, or one of two different red-tailed hawk calls. All squirrels exhibited increased antipredator behavior after hearing increasingly threatening stimuli. Consistent with prior findings in other species with color polymorphism, gray morphs were more likely to escape after hearing a threatening call. A growing body of evidence indicates it is possible to study pleiotropic effects of genes in free living animals.