Aims The aim was to identify the main drivers of aquatic macroinvertebrate species richness in spring-fen habitats (i.e. groundwater seepage wetlands) because these habitats are among the most threatened temperate biodiversity hotspots. Location Isolated spring fens in the western Carpathian Mountains. Methods Assemblages of Tricladida, Clitellata, Mollusca, Ephemeroptera, Plecoptera, Trichoptera and Diptera were sampled at 48 fens distributed evenly along a gradient of water mineral richness and grouped according to habitat specialization and dispersal mode. Several physicochemical parameters that have been reported to be the main drivers of species distribution were recorded for each site, along with radiocarbon measurements of the absolute age of the sites. The numbers of species in the taxonomic groups analysed were modelled as a function of the predictors via multiple linear regressions. Results We achieved a notably higher sampling efficiency than had been used in previous spring aquatic macroinvertebrate studies. In total, we collected and identified 255 species within 331 taxa from > 235,000 individuals. The number of species in all taxonomic groups increased with water discharge but the highest predictive power was obtained with water redox potential, which explained as much as 55.7% of the specialist species richness variance. We found contrasting and systematic differences in the importance of predictors related mainly to the level of species habitat specialization. Species richness of springfen specialists was strongly determined by the main environmental gradient of change in groundwater chemistry, while generalists primarily reflected habitat stability linked to higher water discharge, habitat size and absolute age. Main conclusion Isolated island-like spring fens can harbour unusually species-rich assemblages of aquatic macroinvertebrates, the species richness of which is shaped by contrasting mechanisms dependent mainly on habitat specialization and also partly on dispersal mode. The richness of habitat specialist species seen at calcareous fens indicates their conservation priority.