Trophic partitioning, defining how individuals or populations differ in their resource use, is expected to promote coexistence of sympatric species by reducing resource overlap. We used stable isotope measurements (delta C-13 and delta N-15) to address niche characteristics (delta C-13 and delta N-15 ranges, isotope convex hull and standard ellipse areas, mean nearest neighbour distances and trophic positions) and trophic partitioning of sympatric brown trout (Salmo trutta, n = 110) and European grayling (Thymallus thymallus, n = 63) in two sampling locations of a French river. Aquatic resources predominantly fuelled both species, yet both terrestrial resources (TER) and trophic positions (TP) were higher for trout (similar to 36 +/- 13% TER, 3.6 +/- 0.7 TP) than grayling (similar to 26 +/- 9% TER, 2.7 +/- 0.6 TP) supporting difference in their trophic niches. Isotope analyses showed that trout had a larger isotope niche than grayling suggesting more opportunistic trophic behaviour. Their isotopic overlapping was higher at the upstream site (isotopic nestedness = 0.8 +/- 0.1) than that at the downstream site (isotopic nestedness = 0.4 +/- 0.2). Euclidian distances of stable isotopes and TP for the two species increased with salmonid size, while aquatic resource use decreased with salmonid size. These results demonstrate an increase in isotopic niche partitioning and change in trophic attributes with ontogeny. Our study showed that despite relying on similar resources, these two sympatric salmonids exhibited clear trophic differences that were amplified with ontogeny. The consideration of fish ontogenetic dietary shift would hence be a determinant driver of the trophic niche partitioning for these sympatric salmonids.