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Abstract: The objective of this study was to evaluate how inter-annual changes in the volume of freshwater input and water parameters (salinity, temperature, major dissolved nutrients, seston and chlorophyll a) affect fish assemblages in the Guadiana estuary (South Portugal). During the sampling period (two distinct hydrological years), 56 fish species were identified. Anchovies (Engraulis encrasicolus) and barbells (Barbus species) dominated the abundances in the high inflow year (2001), but Pomatoschistus species were the most important taxa in the low inflow year (2002). Barbells and Portuguese toadfish (Halobatrachus didactylus) dominated the biomass in both years under different inflow conditions, but a reduction in barbells'' biomass occurred during the low inflow year. Multivariate analysis indicated a persistent spatial structuring of the estuarine community for both years and in different seasonal periods. Changes in salinity and seston, which were mainly due to changes in freshwater input, had an important influence on the structure of the fish assemblages. In 2002, the increased salinity in the upper estuary allowed colonization by marine species of an area that usually contains freshwater, decreasing even more the habitat for indigenous freshwater species in the downstream area of the Guadiana River. There was also a decrease in the abundances of planktivorous and omnivorous fishes and an increase in carnivorous fishes during the low inflow year. As fishes in these systems are important regulators of processes in the trophic web, changes in the dominant feeding groups can have consequences on water quality, particularly in relation to the occurrence of plankton blooms. [Copyright &y& Elsevier]