The relative importance of neutral processes and selection in driving rapid trait differentiation is often unclear. Determining which of these contributing factors is more important would allow us to make more accurate predictions regarding how within-species evolution occurs. To study this relationship, venomous snakes have emerged as a convenient system because the link between venom genotype and phenotype is tractable. The present study focuses on the contributions of neutral and adaptive processes to venom evolution in distinct northern and southern populations of the Eastern Diamondback rattlesnake (Crotalus adamanteus) on Jekyll Island in Georgia. To better understand the contributions of these evolutionary drivers, we examine two factors: whether State Road 520 prevents snake dispersal between populations and whether venom phenotype differs between populations. Radiotracking data reveals that State Road 520 may prevent movement of snakes between populations and limit gene flow, thus maintaining or driving the observed population genetic differences. Characterization of venom using reversed-phase high performance liquid chromatography does not show significant differences between venom phenotypes of the two populations. Our results suggest that northern snakes underwent rapid adaptive convergence to acquire a venom phenotype resembling that of southern snakes, and that venom adaptation on Jekyll Island may be repeatable.