This study is the first attempt to assess the distribution patterns of copepod diversity and community structure and their generative processes in the St. Lawrence River (Quebec). Zooplankton samples were collected and environmental data measured during two expeditions of the research vessel “Lampsilis”, conducted during May and August 2006. A total of 52 stations, distributed along 16 transversal transects, were visited in three main biogeographical zones along the longitudinal fluvial-estuary continuum. Upstream, the fluvial section zone (FSZ) includes three freshwater lakes connected by fluvial corridors. Downstream, the fluvial estuary zone (FEZ) is characterized by the presence of fresh and slightly brackish waters, while the estuarine transition zone (ETZ) is characterized by the mixing of fresh and salt waters, high-intensity upwelling currents, and strong tides.We distinguished six different water masses in the St. Lawrence River. To describe the spatial distribution patterns of the main groups of copepods (calanoids, cyclopoids, harpacticoids), we compared copepod diversity and species assemblages among the three biogeographic zones during two different hydroperiods in May (high discharge) and August (low discharge). Different copepod assemblages were observed along the longitudinal river-estuary continuum according to their species richness, abundance, and community structure. The fluvial copepod community greatly differed from the estuarine copepod community. We established the relationship between copepod distribution patterns and the spatial distribution of water masses and their physico-chemical characteristics during each hydroperiod. We showed that the longitudinal discontinuity in water masses and the conductivity-turbidity gradient were the most important drivers, explaining 82% of total variation in copepod community structure during the spring hydroperiod. However, during the summer hydroperiod, water mass distribution and chemical characteristics explained only 37% of the total variation in copepod community structure, indicating that unmeasured biotic factors (such as food resources and predation pressure) may have a stronger influence than hydrologic and abiotic factors during summer.