Cardiolipin is a hallmark phospholipid of mitochondrial membranes. Despite established significance of cardiolipin in supporting respiratory supercomplex organization, a mechanistic understanding of this lipid-protein interaction is still lacking. To address the essential role of cardiolipin in supercomplex organization, we determined cryo-EM structures of a wild-type supercomplex (IV1III2IV1) and a supercomplex (III2IV1) isolated from a cardiolipin-lacking Saccharomyces cerevisiae mutant at 3.2-Å and 3.3-Å resolution, respectively. Phosphatidylglycerol is located in the III2IV1 supercomplex at similar positions as cardiolipin in the wild-type supercomplex. Lipid-protein interactions in the latter are different from that observed for the former that conceivably underlies the reduced stability of supercomplexes in mutant mitochondria. Our studies provide evidence that anionic phospholipids appear to nucleate a phospholipid domain at the interface between the individual complexes, which may contribute to supercomplex stability. Destabilization of supercomplex formation by phosphatidylglycerol has significant implications for Barth Syndrome patients where mitochondrial phosphatidylglycerol levels are significantly elevated.