AbstractTheDrosophilaGenetic Reference Panel (DGRP) serves as a valuable resource to better understand the genetic landscapes underlying quantitative traits. However, such DGRP studies have so far only focused on nuclear genetic variants. To address this, we sequenced the mitochondrial genomes of >170 DGRP lines, identifying 229 variants including 21 indels and 7 frameshifts. We used our mitochondrial variation data to identify 12 genetically distinct mitochondrial haplotypes, thus revealing important population structure at the mitochondrial level. We further examined whether this population structure was reflected on the nuclear genome by screening for the presence of potential mito-nuclear genetic incompatibilities in the form of significant genotype ratio distortions (GRDs) between mitochondrial and nuclear variants. In total, we detected a remarkable 1,845 mito-nuclear GRDs, with the highest enrichment observed in a 40 kb region around the geneSex-lethal(Sxl). Intriguingly, downstream phenotypic analyses did not uncover major fitness effects associated with these GRDs, suggesting that a large number of mito-nuclear GRDs may reflect population structure at the mitochondrial level rather than actual genomic incompatibilities. This is further supported by the GRD landscape showing particular large genomic regions associated with a single mitochondrial haplotype. Next, we explored the functional relevance of the detected mitochondrial haplotypes through an association analysis on a set of 259 assembled, non-correlating DGRP phenotypes. We found multiple significant associations with stress- and metabolism-related phenotypes, including food intake in males. We validated the latter observation by reciprocal swapping of mitochondrial genomes from high food intake DGRP lines to low food intake ones. In conclusion, our study uncovered important mitochondrial population structure and haplotype-specific metabolic variation in the DGRP, thus demonstrating the significance of incorporating mitochondrial haplotypes in geno-phenotype relationship studies.