The island spotted skunk (Spilogale gracilis amphiala) is endemic to the 2 largest California Channel Islands, Santa Cruz and Santa Rosa. Unlike the island fox (Urocyon littoralis) and island subspecies of the deer mouse (Peromyscus maniculatus), the island spotted skunk shows no morphological differentiation between islands and is differentiated only weakly from mainland subspecies, suggesting recent colonization. However, the islands have been isolated from each other and the mainland throughout the Quaternary Period. We used 8 microsatellite loci to investigate the distribution of genetic variation within and among populations of spotted skunks from 8 localities (the 2 islands and 6 mainland localities), representing 4 subspecies. Tissue samples were obtained from 66 fresh specimens collected from 2000 to 2002 and 142 museum specimens collected from 1906 to 1994. Allelic richness and heterozygosity in island spotted skunk populations was approximately 30% lower than that found in mainland localities or subspecies. All localities or subspecies were significantly differentiated (mean F_ST was 0.17 and 0.13 for localities and subspecies, respectively). Contrary to comparisons based on morphological data, genetic differentiation was especially strong between islands and between island and mainland localities or subspecies. Patterns of differentiation suggest that skunks colonized the Channel Islands shortly before rising sea levels separated Santa Cruz and Santa Rosa islands (11,500 years ago). Our results indicate that the taxonomic status of the island spotted skunk should be reconsidered and that both island populations might constitute evolutionarily significant units worthy of conservation.