In this study, we analyse spatial patterns of macrobenthic communities of the Río de la Plata system, and assess the species turnover or beta diversity and its relationship with environmental gradients. Macrobenthic samples and physico-chemical parameters were collected from 20 sampling sites along a transect of 560 km, including the freshwater (FW), estuarine (ES) and marine (MA) sectors. Three main assemblages corresponding to the above mentioned sectors were defined with multivariate analysis (cluster, MDS). In total 134 taxa were recorded, 81 in MA, 33 in FW and 38 in ES, represented mainly by polychaete, mollusc and crustacean species. Depth, salinity and %clay showed the strongest correlation with the observed faunal patterns (ρw=0.62; BIO-ENV analysis). Beta diversity varied between dominant taxonomic groups and was positively correlated with changes in salinity. The high variability in the composition of assemblages was reflected in beta diversity, reaching its highest values at the boundaries between the defined sectors. This study suggests that beta diversity represents a useful tool to define ecological boundaries for benthic communities in the Río de la Plata.