Abstract. Diatoms (Bacillariophyta) are one of the most common and diverse algal groups (ca. 200 000 species, ≈ 10–200 μm, unicellular, eukaryotic). Here we investigate the potential of aerial diatoms (i.e. diatoms nearly exclusively occurring outside water bodies, in wet, moist or temporarily dry places) to infer surface hydrological connectivity between hillslope-riparian-stream (HRS) landscape units during storm runoff events. We present data from the Weierbach catchment (0.45 km², northwestern Luxembourg) that quantify the relative abundance of aerial diatom species on hillslopes and in riparian zones (i.e. surface soils, litter, bryophytes and vegetation) and within streams (i.e. stream water, epilithon and epipelon). We tested the hypothesis that different diatom species assemblages inhabit specific moisture domains of the catchment (i.e. HRS units) and, consequently, the presence of certain species assemblages in the stream during runoff events offers the potential for recording whether there was hydrological connectivity between these domains or not. We found that a higher percentage of aerial diatom species was present in samples collected from the riparian and hillslope zones than inside the stream. However, diatoms were absent on hillslopes covered by dry litter and the quantities of diatoms (in absolute numbers) were small in the rest of hillslope samples. This limits their use for inferring hillslope-riparian zone connectivity. Our results also showed that aerial diatom abundance in the stream increased systematically during all sampled events (n = 11, 2011–2012) in response to incident precipitation and increasing discharge. This transport of aerial diatoms during events suggested a rapid connectivity between the soil surface and the stream. Diatom transport data were compared to two-component hydrograph separation, and end-member mixing analysis (EMMA) using stream water chemistry and stable isotope data. Hillslope overland flow was insignificant during most sampled events. This research suggests that diatoms were likely sourced exclusively from the riparian zone, since it was not only the largest aerial diatom reservoir, but also since soil water from the riparian zone was a major streamflow source during rainfall events under both wet and dry antecedent conditions. In comparison to other tracer methods, diatoms require taxonomy knowledge and a rather large processing time. However, they can provide unequivocal evidence of hydrological connectivity and potentially be used at larger catchment scales.