The Anacapa deer mouse is an endemic subspecies that inhabits Anacapa Island, part of Channel Islands National Park. Anacapa is a chain of three small islands (East, Middle and West). In this study, we used mitochondrial DNA (mtDNA) cytochrome c oxidase subunit II gene (COII) and ten microsatellite loci to evaluate the levels of genetic differentiation and variation in ~1400 Anacapa deer mice sampled before and after a black rat (Rattus rattus) eradication campaign that included trapping, captive holding and reintroduction of deer mice. We also compared levels of genetic variation in Anacapa deer mice to those of a nearby mainland population. Both mitochondrial and microsatellite analyses indicated significant differentiation between Anacapa deer mice and mainland mice, and genetic variability of mainland mice was significantly higher than Anacapa mice even prior to reintroduction. We also found that East, Middle and West Anacapa mice were genetically differentiated from each other, but translocation of mice among islands resulted in the East population becoming less distinct as a result of management. Levels of heterozygosity were similar before and after management. However, numerous private alleles in the founder populations were not observed after reintroduction and shifts in allele frequencies occurred, indicating that the reintroduced populations experienced substantial genetic drift. Surprisingly, two mitochondrial haplotypes observed in an earlier study of Anacapa deer mice were lost in the 20 years prior to the rat eradication program, leaving only a single haplotype in Anacapa deer mice.