The spatial distribution of the subtidal crustacean assemblages of the Guadiana River estuary was studied previous to the building of the Alqueva Dam (the biggest dam in Europe). The differences between an estuarine and marine environment seem to be the main reason responsible for the composition and distribution of the crustaceans along the study zone. The Guadiana estuary has shown high number of species in comparison with other nearby estuaries and this richness seems justified by the scarce influence of pollutants (most of them have shown low or moderate values) and their hydrodynamic and granulometric characteristics (76 species were found, 39 in the estuarine area). A gradient of enrichment and structuring of the assemblages was shown from the upstream to the marine zones and a spatial segregation of species was found along this estuarine environmental gradient, e.g. the amphipod Corophium multisetosum and the isopod Cyathura carinata in the upper estuary; the amphipod Bathyporeia cf. pilosa and the isopods Lekanesphera levii and Saduriella losadoi in the middle estuary; the amphipod Melita hergensis in the mouth; and a high number of species in the marine area. Since the Alqueva Dam will reduce the river discharges and may cause changes in the abiotic characteristics such as granulometry of sediments or salinity, this study establishes a baseline against which a monitoring programme or follow-up studies could measure any significant effects of the dam or related impacts.