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Abstract: In transitional waters the process of defining reference conditions (in the scope of the WFD) must account for the natural great variability of such environments. Therefore, stretches reflecting different physical–chemical and biological conditions throughout the system should be defined in order to correctly establish benthic specific reference conditions. Both salinity and sediment structure are major factors controlling physical–chemical conditions and therefore organisms’ distribution within an estuary. These environmental variables (salinity, sediment grain size composition and organic matter content) patterns were studied in the Mondego estuary and some clear gradients emerged. Also, ecological indices (AMBI, Margalef and Shannon-Wiener) were applied to subtidal benthic communities of the Mondego estuary and, generally, there was not only evidence of a decrease in diversity in the estuary from the downstream section towards its inner parts, but also differences were found between areas of distinct sediment composition. After comparing environmental patterns with biodiversity trends, the information was used to define homogeneous sectors along a temperate estuary in Portugal. In the Mondego estuary six zones, covering the main physical gradients affecting benthic communities, were defined: four in the northern arm and two in southern arm. Zones established will allow future determination of benthic reference conditions adjusted for each of the sectors, according to their characteristics, and consequently the conditions they provide for benthic assemblages settlement. [Copyright &y& Elsevier]