Background: Some populations of Drosophila subobscara (Collin) inhabit geographically separate and ecologically distinct habitats of the central Balkans. Hypothesis: Some of the separate populations were genetically isolated in glacial refugia and have subsequently diverged, leading to high genetic diversity. Methods: We sampled five different D. subobscum populations. Using fragment analysis for 11 microsatellite loci, we used standard diversity parameters (expected heterozygosity, allelic richness, allele size range) to estimate population genetic structure and genetic diversity. Results: We found significant differences in the number of alleles, range of allele sizes, and expected heterozygosity between populations from ecologically dist...