Significance Spatial organization of mitochondria is vital for cellular function. In many specialized cell types, mitochondria are immobilized at specific subcellular loci through interactions with the cytoskeleton. One of the most striking mitochondrial configurations occurs in mammalian sperm, where mitochondria wrap around the flagellum. Malformation of the mitochondrial sheath causes infertility, but the molecular structures underlying this intricate arrangement are unknown. Here, we analyzed the mitochondrial sheath in sperm from three mammalian species. We find that although mitochondrial dimensions and cristae architecture vary across species, molecular assemblies mediating intermitochondria and mitochondria–cytoskeleton interactions are conserved. These findings yield important insight into sperm physiology and evolution and are relevant for other polarized cell types, such as muscles, neurons, photoreceptors, and hair cells.