The Papahānaumokuākea Marine National Monument surrounding the Northwestern Hawaiian Islands (NWHI) is the largest marine protected area in the United States and among the largest on Earth. The geographic isolation of this region has produced a unique biodiversity that is marked by particularly high levels of endemism. Previous surveys have revealed that on average 21% of reef fish species in the NWHI are endemic to the Hawaiian Archipelago. However, these endemism estimates are based on scuba surveys in shallow waters (<30 m). In areas with high water clarity like Hawaii, coral reefs can extend to depths exceeding 150 m. We conducted 52 mixed-gas dives and recorded a total of 179 reef fish species at mesophotic coral reef depths (30–90 m) across the NWHI. About 46% of encountered reef fishes in the NWHI are endemic to Hawaii, a value that is 16%–24% higher than previous shallow-water (<30 m) surveys in the NWHI, as well as nearly two-fold higher than in any other tropical region. As noted previously for shallow reefs in the NWHI, endemism appears to increase with latitude, as relative abundances of endemic reef fishes on mesophotic reefs ranged from 16% at the southernmost end of the NWHI, to upwards of 92% at the northernmost end of the NWHI. This unprecedented rate of endemism indicates that mesophotic reefs in the NWHI are reservoirs of biodiversity, and further underscores the need for protection of this area.