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Mesophotic coral ecosystems (MCEs) are tropical reefs found at depths of ~30–150 m, below the region most heavily impacted by heat stress and other disturbances. Hence, MCEs may serve as potential refugia for threatened shallow reefs, but they also harbour depth‐endemic fauna distinct from shallow reefs. Previous studies have characterized biodiversity patterns along depth gradients, but focussed primarily on conspicuous taxa (fishes, corals, etc.). Environmental DNA (eDNA) metabarcoding offers a more holistic approach to assess biodiversity patterns across the tree of life. Here, we use three metabarcoding assays targeting fishes (16S rRNA), eukaryotes (18S rDNA) and metazoans (COI) to assess biodiversity change from the surface to ~90 m depth across 15‐m intervals at three sites within the Hawaiian Archipelago. We observed significant community differences between most depth zones, with …