Corals at the lower limits of mesophotic habitats are likely to have unique photosynthetic adaptations that allow them to persist and dominate in these extreme low light ecosystems. We examined the host–symbiont relationships from the dominant coral genus Leptoseris in mesophotic environments from Hawai'i collected by submersibles across a depth gradient of 65–125 m. Coral and Symbiodinium genotypes were compared with three distinct molecular markers including coral (COX1–1-rRNA intron) and Symbiodinium (COI) mitochondrial markers and nuclear ITS2. The phylogenetic reconstruction clearly resolved five Leptoseris species, including one species (Leptoseris hawaiiensis) exclusively found in deeper habitats (115–125 m). The Symbiodinium mitochondrial marker resolved three unambiguous haplotypes in clade C, which were found at significantly different frequencies between host species and depths, with one haplotype exclusively found at the lower mesophotic extremes (95–125 m). These patterns of host–symbiont depth specialization indicate that there are limits to connectivity between upper and lower mesophotic zones, suggesting that niche specialization plays a critical role in host–symbiont evolution at mesophotic extremes.