Sequence data from a 597–base pair region of the mitochondrial DNA control region and data for 19 presumed allozyme loci were used to examine genetic structure and to detect gene flow among populations of southern red-backed voles (Myodes gapperi, formerly known as Clethrionomys gapperi) inhabiting spruce–fir “sky-islands” in the southern Appalachians. Allozyme data showed a significant heterozygote deficiency for most populations. Average individual heterozygosity varied among populations from 1.75% to 16.78% (X̄ ± SE = 8.63% ± 2.03%). F_ST values between any 2 sites ranged from 0.100 to 0.517 (X̄ ± SE = 0.296 ± 0.026), with a significant metapopulation F_ST value = 0.599. Nei's genetic distances (based on allozyme data) among spruce–fir habitats ranged from 0.037 to 0.310 (X̄ ± SE = 0.139 ± 0.079). Analysis of molecular variance permitted determination of the extent of geographic subdivision of mitochondrial haplotypes for 3 hierarchically organized sampling regimes: 14 high-altitude sample sites; 7 spruce–fir islands within which the sample sites are located; and whether sample sites were located south or north of the dominant, low-altitude barrier, the French Broad River. Genetic variation among groups and Φ_ST values were highly significant for all sampling regimes, indicating significant population structure at all hierarchical levels tested. Mantel's general regression test indicated that gene flow and genetic relationships fit an isolation-by-distance model. Since the Pleistocene, gene flow within the metapopulation of M. gapperi appears to have declined as a result of increasing distances between habitat islands and because of population bottlenecks.