Avian predation is a primary cause of mortality for Spilogale putorius (Eastern Spotted Skunk) and is generally attributed to Bubo virginianus (Great Horned Owl). We report the first confirmed avian predation of an Eastern Spotted Skunk by a Strix varia (Barred Owl). The radio-collar of an adult female Eastern Spotted Skunk was recovered alongside an owl pellet containing Eastern Spotted Skunk remains in eastern West Virginia. DNA sampled from the surface of the pellet was amplified, sequenced, and compared to known Barred Owl and Great Horned Owl sequences. Differences in 2 single nucleotide polymorphisms confirmed a Barred Owl produced the pellet. Our findings show Barred Owls are at least occasionally an avian predator of this species in eastern forest ecosystems.