The strength and arrangement of movement barriers can impact the connectivity among habitat patches. Anthropogenic barriers (e.g. roads) are a source of habitat fragmentation that can disrupt these resource networks and can have an influence on the spatial genetic structure of populations. Using microsatellite data, we evaluated whether observed genetic structure of moose (Alces alces) populations were associated with human activities (e.g. roads) in the urban habitat of Anchorage and rural habitat on the Kenai Peninsula, Alaska. We found evidence of a recent genetic subdivision among moose in Anchorage that corresponds to a major highway and associated infrastructure. This subdivision is most likely due to restrictions in gene flow due to alterations to the highway (e.g. moose-resistant fencing with one-way gates) and a significant increase in traffic volume over the past 30 years; genetic subdivision was not detected on the Kenai Peninsula in an area not bisected by a major highway. This study illustrates that anthropogenic barriers can substructure wildlife populations within a few generations and highlights the value of genetic assessments to determine the effects on connectivity among habitat patches in conjunction with behavioral and ecological data.