The presence of persistent fish assemblage areas in estuaries can result from spatially structured environmental gradients, which determine a pattern of biological zonation. In the Rı́o de la Plata estuary (36°S, 56°W), those assemblage areas were defined for summer, autumn, and spring of 1996. Cluster analysis identified groups of sampling stations with a similar species composition for each season. Our results identified three fish assemblage areas along the main axis of the estuary. These assemblages are persistent in specific composition and correspond to particular oceanographic environments throughout the year. The boundaries of the assemblage areas are associated with the highest horizontal gradients of salinity. Geographic variations of the boundaries, indicating contraction or expansion of fish assemblage areas, were associated with the spatial estuarine dynamics. The fish assemblage areas reflect the local pattern of habitat heterogeneity. This suggests that for large estuaries, the fish assemblage areas are indicators of this spatial scale of habitat heterogeneity, as defined by salinity and stratification of water column.