The genetic structure of seven populations of Drosophila subobscura from different locations on Majorca and Minorca (Balearic Islands, Spain) was studied using two types of markers: allozyme and mitochondrial DNA restriction analyses. Both markers showed congruent results. In the allozyme data, when the Acph-1 locus was excluded from the joint FST statistics, only three out of 21 comparisons were statistically significant, lending support to the hypothesis of low genetic differentiation. The mtDNA restriction analyses showed two haplotypes at a high frequency (more than 40% each), irrespective of the location considered, and a number of endemic haplotypes at very low frequencies (not higher than 2% each). The analyses of the genetic structure yielded a pattern similar to the allozymes. The cytonuclear disequilibrium analyses showed the difficulty of detecting cytonuclear associations in natural populations because they are mainly transient.