Trophic-niche differentiation is often cited as a main factor in structuring zooplankton assemblages, although field evidence for this is rarely presented. The study was based on a survey of 29 Pyrenean lakes with altitudes ranging between 1,875 and 2,990 m carried out during July and August 2000. Because of the oligotrophic nature of these lakes, we aimed to confirm that food partitioning is a major factor in shaping zooplankton assemblages. We analysed the amino acid composition of six cladocera and seven copepod species. A discriminant analysis showed that each species could be distinguished according to its amino acid composition. A negative relationship between amino acid differentiation and co-occurrence among the cladocera and cyclopoid copepod was observed. In contrast, calanoids did not show any relationship and were characterised by a high amino acid differentiation between species. As the differences in the amino acid composition among zooplankton species indicate distinct food sources, the relationship found indicates that trophic-niche differentiation plays a key role in determining the assemblage of these zooplankton communities. Therefore exploitative competition, either at present or in the past by driving co-evolutionary histories, has been a significant factor in structuring the cladocera and cyclopoid communities in these oligotrophic lakes.