The connecting channels linking the Laurentian Great Lakes provide important migration routes, spawning grounds, and nursery habitat for fish, but their role as conduits between lakes for zooplankton is less understood. To address this knowledge gap in the St. Clair–Detroit River System (SCDRS), a comprehensive survey of crustacean zooplankton was performed in both riverine and lacustrine habitats from spring to fall 2014, providing the first system-wide assessment of zooplankton in the SCDRS. Zooplankton density and biomass were greatest in northern reaches of the system (southern Lake Huron and the St. Clair River) and decreased downstream towards Lake Erie. The composition of zooplankton also changed moving downstream, transitioning from a community dominated by calanoid copepods, to more cyclopoids and cladocerans in the Detroit River, and to cladocerans dominant in western Lake Erie. Coincidentally, species richness increased as sampling progressed downstream, and we estimated that our single-year sampling regime identified ~88% of potential taxa. Other species assemblages have responded positively to recent water quality and habitat restoration efforts in the SCDRS, and this survey of the zooplankton community provides benchmark information necessary to assess its response to continued recovery. In addition, information regarding the lower trophic levels of the system is integral to understanding recruitment of ecologically and economically valuable fish species targeted for recovery in the SCDRS.