In this study we explored 54 isolated spring fens across the eastern Czech Republic and Slovakia. These fens varied in calcium and magnesium concentrations, forming a sharp and well defined environmental gradient running from calcium-poor acidic fens to extremely calcium-rich tufa-forming fens. We found that the main changes in clitellate species composition were controlled by this gradient, and/or total organic carbon content, over a wide area, including fen sites differing in other environmental conditions and historical development. However, this pattern was weakened in sites with a high organic matter content,which represented a second driver of change in assemblage composition along with water temperature. Three main types of fens were determined using cluster analysis based on clitellate assemblage composition. However, only the first type, which included tufa-forming fens, was found to fit with thepreviously established spring fen types based on vegetation (i.e. extremely mineral-rich fens with a tufa, brown-moss mineral-rich fens, mineral-rich Sphagnum fens and mineral-poor Sphagnum fens). The second clitellate type included sites with low temperatures and occasional desiccation, while the third typewas characterised by high temperatures and trophy. Using eight environmental predictors, we were able to significantly explain changes in the population abundances of all 12 common species (i.e. recorded at 15-plus sites). The results from individual species modelling also suggests that an increase in organic matter content can trigger compositional shifts towards assemblages of common eurytopic tubificid species. Thus, human-induced eutrophication and negative changes in spring fen hydrology, mainly drying up, can represent a serious threat for species-specific assemblages of aquatic clitellates, especially at alkaline sites due to their isolated and spatially limited nature.