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Even though alderflies, as well as many other aquatic macroinvertebrates, are widely used as indicators of freshwater quality, the knowledge of their distribution, biology and ecology in karst freshwater habitats is still very scarce. Aiming to fill this gap, we investigated alderfly assemblages along an oligotrophic karst hydrosystem. Sampling was carried out on a monthly basis over a one-year period (2007/2008) in different types of freshwater habitats and microhabitats. Our goals were to determine population aspects, habitat and microhabitat preferences of alderflies as well as their relation to environmental factors. Due to the specificity of karst freshwater habitats, alderflies were rather rare in the studied hydrosystem. Yet, their fauna was diverse with four identified species, where Sialis fuliginosa Pictet, 1836 was the most widespread one. Sialis sordida (Klingstedt, 1932) was recorded for the first time in the Balkan Peninsula being the rarest species in our study. Substrate type and water velocity showed as key environmental parameters in determining alderfly assemblages. Alderflies were mostly recorded from a canyon type mountainous stream, and silt with leaf litter, respectively. They were not recorded at sites characterized by predominately solid stones (i.e. cobbles) and mosses, and with intermittent water flow. Our results strongly indicate that alderflies could be used in future conservation and protection management procedures in karst freshwater habitats. [ABSTRACT FROM AUTHOR]