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Fragmentation and interconnection of water bodies cause severe changes in the natural flow regime of continental water systems. However, assessments of these impacts on the freshwater fish distribution are still scarce. We investigated whether an artificial flow regime generated by discharges from an aqueduct would promote a longitudinal environmental gradient in an off-river reservoir. We tested the hypothesis that the incoming water from aqueduct discharges promotes environmental gradient between lotic and lentic conditions. We expect that environmental changes and the artificial flow regime promote the structuring of fish assemblage and that the lotic-lentic transition areas present the highest fish species richness. A longitudinal gradient in environmental characteristics with differing fish assemblage structure was found. Temporal changes in the fish assemblages occurred only in areas near the discharge of the aqueduct. The highest fish richness and diversity were recorded in the transition zone during the wet season, associated with the greater environmental heterogeneity of this ecotone, whereas the lowest was found in the lotic zone, probably associated with the altered flow regime. The greatest abundance was recorded in the upper lentic zone in the wet season, whereas the lowest was found in the lower lentic zone in both seasons. Our results corroborate the hypothesis that the artificial flow regime, modulated by the seasonality of discharges and the influence of environmental variables, promotes longitudinal gradients in reservoirs when rivers are not present. The findings are relevant in a scenario where ever more interconnections of aquatic bodies have been diffused around the world.