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Stoneflies are among the most sensitive aquatic insect taxa and therefore arguably the best indicator of the excellent, i.e. pristine, ecological status of surface streams. Karst habitats are one of the most exciting freshwater habitats in terms of biological-geological interplay. They, in turn, support a biodiversity far superior to surrounding freshwater habitats and hence these habitats are designated as biodiversity hotspots. Our study deals with both of these crucial ecological players. We studied stonefly assemblages, their microhabitat preferences and emergence patterns along a karst oligotrophic hydrosystem. The sampling was conducted monthly from March 2007 to December 2008 using pyramid-type emergence traps set in various habitats and associated microhabitats (e.g. springs, rivers, streams, tufa barriers × moss, angiosperm, cobble, sand, silt substrates). Favorable environmental conditions, such as a wide range of karst habitat types with low water temperature and high oxygen concentration, resulted in high stonefly species richness (31 recorded species). Water temperature and pH had the highest influence on stonefly assemblages. Species richness and diversity decreased in a downstream direction. We recorded a longitudinal shift from crenal-epirhithral to epirhithral-metarhithral assemblages with some hyporhithral and potamal elements. Upstream sites were dominated by shredders, while downstream sites had a higher proportion of gatherers-collectors. Several species showed a significant preference for a specific microhabitat type in accordance with their feeding strategies and food availability. The majority of recorded species exhibited univoltine life cycles slow or fast.