We defined mesozooplankton biogeography in the North American Arctic to elucidate drivers of biodiversity, community structure, and biomass of this key component of the Arctic marine ecosystem. A multivariate analysis identified four mesozooplankton assemblages: Arctic-oceanic, Arctic-shelf, Coastal-Hudson, and Labrador Sea. Bathymetry was a major driver of the distribution of these assemblages. In shallow waters, Cirripedia and the copepod Pseudocalanus spp. dominated the Coastal-Hudson and Arctic-shelf assemblages, which showed low species richness (19) and biomass (0.28 and 1.49 g C m−2, respectively). The Arctic-oceanic assemblage occupied the entire North American Arctic, except for shallow breaks in the Canadian Arctic Archipelago downstream of sills blocking the Atlantic Water layer circulation below a depth of 200 m. This assemblage showed high copepod biomass (4.74 g C m−2) with a high share of Calanus hyperboreus, C. glacialis, and Metridia longa. In habitats below 200-m depth, C. hyperboreus represented 68% of the copepod biomass, underscoring its role as a keystone species in this ecosystem. Strong numerical representation by the boreal-Atlantic C. finmarchicus and Oithona atlantica stressed the strong Atlantic influence on the subarctic Labrador Sea assemblage on the northwestern Labrador Sea slope. The mixed Arctic-Atlantic composition of the Labrador Sea mesozooplankton resulted in high species richness (58) and biomass (5.73 g C m−2). The low abundance of Atlantic and Pacific taxa in the areas influenced by Arctic currents did not alter the Arctic status of the Arctic-oceanic, Arctic-shelf, and Coastal-Hudson assemblages. This study identifies hotspots of mesozooplankton biomass and diversity in Central Amundsen Gulf, Lancaster Sound, North Water Polynya and Baffin Bay, known for their high biological productivity and concentrations of vertebrate predators. The continental-scale zooplankton mapping furthers our understanding of the importance of bathymetry and ocean circulation for ecological connectivity in a vast and complex portion of the Arctic marine ecosystem.