The Wadden Sea is a shallow coastal region, with a large area of sedimentary tidal flats that extends from The Netherlands to Denmark and has been declared a site of international importance in the Dutch and German parts (Ramsar status and UNESCO World Heritage Site). Benthic macrofauna are central to the ecosystem functioning of this area, as they recycle nutrients, decompose organic matter and are an important food source for many secondary consumers, like fish and waterbirds. Due to the environmental gradients characteristic of estuarine systems, it is expected that changes in assemblage composition will be observed across the physical and environmental gradients of the Wadden Sea. First, we explored the spatial variation in assemblage composition of benthic macrofauna across the intertidal part of the Dutch Wadden Sea using 3. years of biomass data. Then, we identified the relative importance of six environmental variables for explaining and predicting changes in assemblage composition across the intertidal areas of the Wadden Sea using generalised dissimilarity modelling (GDM). In accordance with the environmental gradients across this system, the biomass contributed by a few common species differed from west to east and were distinct in the Dollard. In the west, bivalves Mya arenaria, Cerastoderma edule and Ensis directus contributed a relatively large and equal share of the total biomass, whereas C. edule contributed the sole largest share of the total biomass towards the east. The polychaete Alitta succinea became a large share of the total biomass in the upper Ems and in the Dollard estuary, but contributed little elsewhere. Similar to the observed differences in species composition, the spatial patterns in assemblage composition, as predicted by the GDM models, identified the Dollard as distinct and that the prevalence of assemblage types in the west differed to the east. Median grain size, followed by microphytobenthic biomass, and exposure time were the most important variables describing differences in assemblage composition. That the Wadden Sea forms a heterogeneous landscape where assemblage composition varies across multiple gradients has repercussions for management and monitoring. © 2013 Elsevier B.V.