Nearshore oceanic archipelagos are natural laboratories that could provide valuable insight into the role of evolutionary processes such as founder effects and incipient speciation in biotic conservation. The Alexander Archipelago of Southeast Alaska is an example of such a complex, yet few biological investigations have been conducted. For the past 50 years, the region has experienced intense anthropogenic disturbances (particularly timber harvesting), causing habitat fragmentation and potential disruption of biotic communities. As part of a series of studies of mammals endemic to Southeast Alaska, we examined mitochondrial DNA sequences from 118 flying squirrels to investigate genetic diversity across Southeast Alaska. Mitochondrial sequence divergence corroborates the subspecific designation of the endemic Prince of Wales flying squirrel (Glaucomys sabrinus griseifrons). This island lineage exhibits severely reduced genetic variation and may be the result of an early Holocene founder event. Nearly all of the animals we examined on Prince of Wales Island and ten islands to the west had identical cytochrome b (52 of 53) and control region (21 of 21) sequences. In contrast, substantial polymorphism and little genetic structuring were found in comparable populations on the mainland of Southeast and Interior Alaska. Because flying squirrels in the Pacific Northwest are associated with old-growth forest, forest-use plans should aim to conserve this unique lineage of island squirrels.