Island spotted skunks (Spilogale gracilis amphiala) are a rare subspecies endemic to the California Channel Islands, currently extant on Santa Cruz and Santa Rosa islands. How and when skunks arrived on the islands is unknown, hindering decision-making about their taxonomic status and conservation priority. We investigated these questions by sequencing the complete mitochondrial genomes of 55 skunks from the two islands and mainland (California and Arizona) and examining phylogenetic patterns and estimations of isolation times among populations. Island spotted skunks grouped in a single monophyletic clade distinct from mainland spotted skunks. A haplotype network analysis had the most recent common ancestral haplotype sampled from an individual on Santa Rosa, suggesting both islands were colonized by a single matriline. Additionally, no haplotypes were shared between skunk populations on the two islands. These patterns imply that both island populations were derived from a common ancestral population shortly after establishment and have remained isolated from each other ever since. Together with divergence estimates from three methods, this topology is consistent with colonization of the super-island, Santarosae, by a single ancestral population of spotted skunks in the early Holocene, followed by divergence as the sea level rose and split Santarosae into Santa Cruz and Santa Rosa islands 9,400–9,700 years ago. Such a scenario of colonization could be explained either by rafting or one-time transport by Native Americans. Given their distinct evolutionary history, high levels of endemism, and current population status, island spotted skunks may warrant management as distinct evolutionarily significant units.