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Niche partitioning studies are essential to understand the mechanisms that allow ecologically similar species to coexist. The Rhone streber (Zingel asper) and the European bullhead (Cottus gobio) are both benthic riverine fishes that consume macroinvertebrates. Both species are protected under European legislature. We focused on trophic niche partitioning between these species, as sufficient access to trophic resources is an important requirement for long-term coexistence. We used a combination of faeces metabarcoding dietary data and prey community data to evaluate dietary overlap, trophic niche variation and dietary preferences. Sampling was performed in three sites along the Durance River (France). Both species consumed a wide range of macroinvertebrates but neither C. gobio nor Z. asper selected the most abundant taxa (chironomids and simuliids). Both species selected larger mayfly (Baetis and Ecdyonurus) and caddisfly (Hydropsyche) taxa. Dietary overlap was generally high but declined when large mayflies were rare. Contrasting secondary prey preferences and trophic niche variation may allow these species to exploit the same preferred mayflies in periods of abundance and to avoid competition during resource scarcity. We propose that dietary partitioning may support the coexistence of these two ecologically similar, benthic predators.