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According to ecological theory, two species cannot occupy the same niche. Using nitrogen isotope analyses (δ15N) of amino acids, we tested the extent to which two sympatric deposit‐feeding amphipods, Monoporeia affinis and Pontoporeia femorata, partition their trophic resources. We found that trophic position (TP) and resynthesis index (∑V; a proxy for degradation status of ingested material prior to assimilation by the consumer) differ between species. The surface‐feeding M. affinis had higher TP and intermediate ∑V, both pointing to a large contribution of metazoans in its diet. P. femorata, which feeds in the subsurface layers, had lower TP and a bimodal distribution of the ∑V values, supporting previous experimental evidence of a larger feeding niche. We also evaluated whether TP and ∑V values have consequences for amphipod fecundity and embryo viability and found that embryo viability in M. affinis was negatively linked to TP. Our results indicate that the amino acid‐δ15N data paired with information about reproductive status are useful for detecting differences in the trophic ecology of sympatric amphipods. [ABSTRACT FROM AUTHOR]