Drosophila obscura is a very common fruit fly inhabiting European forests. This species has a large number of mitochondrial haplotypes of Cyt b gene. We used experimental lines of D. obscura to test the adaptive significance of intra-population variability of the mitochondrial genome (mtDNA) and selective forces that maintain it. We chose three isofemale lines with distinct mitochondrial haplotypes of Cyt b gene from each of the two populations sampled in Serbia. Using backcrossing, we created nine experimental lines for each population with all combinations of mtDNA haplotypes and nuclear genetic backgrounds (nuDNA). Individuals of both sexes were tested separately for desiccation resistance at two temperatures. Cox proportional hazards model, with four factors: mtDNA, nuDNA, sex and temperature was used to analyze the survival data. In some comparisons we noticed significant effect of mtDNA on desiccation resistance, while all of them showed significant effect of interaction between mitochondrial and nuclear genome. Temperature in interaction with mtDNA or mito-nuclear genotype more frequently showed significant effect on desiccation resistance compared to sex in interaction with mtDNA or mito-nuclear genotype. Our result show adaptive significance of intra-population variation of mtDNA and importance of interactions between mitochondrial and nuclear genome on fitness. Temperature specific mito-nuclear interaction rather than sex-specific selection on mito-nuclear genotypes maintains mtDNA variability in this model species.