Fourteen intertidal sites from the eastern mid-region of the Tagus estuary were considered in order to study their macrobenthic assemblages in relation to environmental factors. The salinity gradient was displayed in an ordination of the assemblages by non-metric multidimensional scaling (NMDS). However, salinity was not found to be a significant environmental factor to explain the distribution of the assemblages. Conversely, a lateral gradient characterized three clusters of sites identified in relation to their outer, intermediate and inner location also related to organic matter content, oxygen concentration and redox potential values. Although the hydrodynamic factor was not investigated it is thought to be an important environmental factor for characterizing the lateral gradient. As a general trend, there was a transition between the groups of assemblages from the lower estuary towards the inner and upstream locations. The polychaetes Lanice conchilega and Hediste diversicolor and the gastropod Hydrobia ulvae were the characteristic species in the outer, inner and intermediate groups of sites, respectively. It was concluded that the lateral gradient is the main environmental driver that explains the distribution of the intertidal macrobenthic assemblages in the Tagus estuary when the salinity gradient is not find to be a significant environmental factor.