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Although the Segura River basin is located in one of Europe’s most arid regions, it features a wide variety of aquatic ecosystems, some of which are rare within the European continent. Assemblages of aquatic Hemiptera and their indicator species in the Segura River basin, as well as the key environmental factors that determine their distribution, were evaluated in this study. Between 1980 and 2010, a total of 38 species of aquatic Hemiptera were collected in 402 sites that have been classified into 12 types of habitats. Aquatic Hemiptera were well-represented among the different habitats and were widely distributed across the entire study area. Relationships between community structure and environmental variables were evaluated using multivariate analyses, including non-parametric multidimensional scaling (NMDS), principal components analysis (PCA) and distance-based redundancy analysis (dbRDA). Results revealed that the distribution of aquatic Hemiptera was influenced primarily by an environmental gradient from lode and freshwater headwater environments to lentic and highly-mineralised waters in lower river sections. Hence, the lotic/lentic character of the habitat and its conductivity were the most important factors shaping the spatial distribution of the aquatic Hemiptera in the Segura River basin. Additionally, an indicator species analysis (IndVal) revealed four aquatic Hemiptera assemblage types: one was related with lotic headwater environments, a second was associated with rivers and reservoirs, a third was found primarily in lotic saline environments and a fourth transitional assemblage type was associated with microhabitat availability and included species with a widespread distribution. Defining Hemiptera assemblage types statistically, however, was difficult due to the widespread distribution of species caused by high dispersion capability and low microhabitat requirements.