Diet information of organisms was traditionally acquired by the use of lethal techniques (gut content or muscle δ13C and δ15N isotopic ratios). An increase in the number of isotopic ratio studies and the vulnerability of some species have led to increased use of non-lethal methodologies for conservation purposes. In the present work we have compared the δ13C and δ15N isotopic signals of muscle and scales of three different fish species in order to test fish-scale sampling as a non-lethal technique in fish trophodynamics. A positive correlation was found between muscle and scales in Argyrosomus regius and Xyrichtys novacula, while Dentex dentex showed no correlation due to the small length range of this species. The isotopic offset (Δ13C and Δ15N) between muscle and scales was significantly different among species (analysis of variance (ANOVA), p < 0.001) with Tukey's post-hoc HSD indicating a tissue offset difference (p < 0.001) for both stable isotopes in all species, except for Δ13C between A. regius and X. novacula and for Δ15N between D. dentex and X. novacula (p > 0.05). Mean δ13C and δ15N values between species showed significant differences (paired t-test, p < 0.01) between muscle and scale with an enrichment for δ13C and a depletion for δ15N. Spatial differences were found in the Δ13C of X. novacula between the studied locations, while no differences were found for Δ15N, indicating that non-geographical differences should be considered in the application of scales instead of muscle for 15N, while for 13C differences in the geographical isotopic offset should be considered. Copyright © 2009 John Wiley & Sons, Ltd.