Timber Rattlesnakes, Crotalus horridus, are an endangered species in New Jersey, where high human population density places extraordinary pressures on natural populations. Disjunct populations are found in both the northern part of the state and a few scattered locations in the southern Pine Barrens. The Pine Barrens populations were previously shown to have reduced levels of genetic diversity compared to other populations in the Mid-Atlantic region. In this study, we used microsatellite DNA to examine the remaining six known C. horridus populations in the Pine Barrens and two in the northern part of the state. Bayesian analysis, genetic distance analysis, and factorial component analysis were performed, and the results indicated that the sampled populations represented four genetic subgroups, the two northern populations comprised one subgroup and the remaining six Pine Barrens populations comprised the other subgroups. Although there was evidence for isolation by distance among the Pine Barrens populations, geographic distance alone did not account for a substantial amount of among-group variation. The combined analyses implicated paved roads as primary isolating barriers between populations. Given their geographic isolation and reduced levels of genetic diversity, intensive efforts may be required to ensure the persistence of the behaviorally and genetically unique C. horridus populations of the New Jersey Pine Barrens.