This study assesses the use of stable isotopes (δ 13 C and δ 15 N) and trace elements in gills (G), liver (L), and muscle (M) of the most commercially important migratory fish from the La Plata Basin, Prochilodus lineatus, as habitat proxies. With this purpose, fish and sediment (S) samples from the Paran a River and Río de la Plata Estuary were analyzed. Results showed differences in the stable isotopic ratios in fish tissue and sediments between the river and the estuary. In particular, δ 15 N values of the estuary samples were significantly lower (G = 7.38‰; L = 6.60‰; M = 8.61‰ and S = 4.36‰) than those from the river (G = 11.30-11.53‰; L = 11.03-12.35‰; M = 11.27-11.31‰, and S = 6.78-7.72‰), which could denote a shift in the isotopic baseline between these environments. Quadratic discriminant analysis effectively discriminates between sampling sites with high classification rates (L = 97.5%; G = 90%, and M = 82.5%), suggesting that combining stable isotopes and trace elements is useful to discriminate between fish populations. The liver was the most suitable tissue as a habitat indicator, using mainly δ 15 N, δ 13 C, Cr, and Hg as natural markers, and capable of discriminating between sites with a relatively high geographic resolution. Due to the spatial isotopic heterogeneity found in the La Plata Basin, these natural markers could be useful for studying other aquatic organisms' habitat use.