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Information on the biodiversity of high altitude lakes in the Stelvio National Park was scarce and fragmentary, in most cases limited to a few studies on a single biological issue. To fill this gap, a multidisciplinary research program was established in 2011 to investigate macroinvertebrates, diatoms, and water chemistry in 8 high altitude lakes within the boundaries of the Park (Rhaetian Alps, Eastern Alps). The results of this study were compared with data on biological assemblages and chemical parameters of Alpine lakes in the Pennine-Lepontine Alps (Western Alps), to evaluate the role of local drivers with respect to regional ones. This comparison was possible thanks to the adoption of standardized sampling methodologies developed since the ’90s by the National Research Council-Water Research Institute (Verbania), in collaboration with several European Research centers. Despite located in a restricted geographical area, the lakes of the Stelvio National Park showed a high variability of chemical composition, and of sensitivity to acidification, lower than that of the Pennine-Lepontine Alpine lakes. Macroinvertebrate and diatom taxa were ubiquitous and frequent along the Alps, and mainly represented by cold-stenothermal species. Richness, Shannon, Simpson, and Pielou indices applied to phyto- and zoobenthos highlighted significantly lower values in Stelvio National Park lakes than in those of Pennine-Lepontine for macroinvertebrates, while no significant differences were found for diatoms. Two groups of lakes were identified by Cluster Analysis, mainly on the basis of major ion concentrations. Canonical Correspondence Analysis showed that the macroinvertebrate assemblage of the lakes studied is driven mainly by altitude and lake surface, and, to a lesser extent, by nutrient content. On the contrary, pH and acid-related variables played a secondary role for diatoms, while nutrients and, more in general, ionic content had significant effects on their species composition. Overall, the results of this first investigation showed that the high elevation of these lakes affects their macroinvertebrate assemblages, while their diatom communities are comparable throughout the Alps.