Spring fens in the Western Carpathians represent groundwater-fed micro-wetlands variable in their water chemistry, ranging from calcium-poor acidic fens with a tendency towards ombrotrophy to extremely calcium-rich tufa-forming fens. These highly vulnerable habitats host specific assemblages of aquatic annelids capable to inhabit both purely aquatic mesohabitats and water-logged soil mesohabitats. To explore the ability of individual species to penetrate into water-logged substratum, we studied the vertical distribution of aquatic annelids in the subsurface interstitial zone (0–15 cm depth). Twenty seven spring fens in the Western Carpathians situated within the Czech and Slovak Republics were sampled in spring and autumn of either 2015 or 2016. We explored sites covering a major environmental gradient: 9 extremely mineral-rich fens with tufa formation, 8 mineral-rich fens without tufa formation, 5 mineral-rich Sphagnum fens and 5 mineral-poor Sphagnum fens. Five cores (17 cm2 each, 15 cm depth) were taken from water-logged mesohabitats (but without a surface water layer) at each site. Each core was subdivided into 3-cm layers. Annelids were extracted using the wet funnel method (without heating) and specimens were identified alive. In total, 1,135 individuals of aquatic Clitellata and 7,926 of Aeolosomatidae were collected from spring samples; autumn samples are being processed. The highest numbers of aquatic annelids were recorded in mineral-poor Sphagnum fens, the lowest in mineral-rich fens with tufa. The first 3 cm of substratum were inhabited by 54% of clitellates and 47% of aeolosomatids; their numbers decreased almost linearly with depth. Median depths at which individual species occurred varied from 1.9 to 10 cm, but most species preferred the upper 3 cm. Taking into consideration annelid life cycles, we expect seasonal differences in vertical distribution. Preliminary results show that of 35 aquatic species previously reported from aquatic habitats (pools, spring brooks) of the same set of sampling sites, 18 were also recorded in the subsurface interstitial zone. These species seem to be well pre-adapted to survive a decrease of the water-table, which is the major threat to spring fen organisms. The study was supported by GA15-15548S and MUNI/A/1325/2015.