The nature and extent of mitochondrial DNA variation in a population and how it affects traits is poorly understood. Here we resequence the mitochondrial genomes of 169 Drosophila Genetic Reference Panel lines, identifying 231 variants that stratify along 12 mitochondrial haplotypes. We identify 1,845 cases of mitonuclear allelic imbalances, thus implying that mitochondrial haplotypes are reflected in the nuclear genome. However, no major fitness effects are associated with mitonuclear imbalance, suggesting that such imbalances reflect population structure at the mitochondrial level rather than genomic incompatibilities. Although mitochondrial haplotypes have no direct impact on mitochondrial respiration, some haplotypes are associated with stress- and metabolism-related phenotypes, including food intake in males. Finally, through reciprocal swapping of mitochondrial genomes, we demonstrate that a mitochondrial haplotype associated with high food intake can rescue a low food intake phenotype. Together, our findings provide new insight into population structure at the mitochondrial level and point to the importance of incorporating mitochondrial haplotypes in genotype-phenotype relationship studies.