In this study we test the potential of stable isotope analysis to reveal wintering origins of waders mixing at stopover sites, using the dunlin Calidris alpina as a case study. We determined stable carbon (δ13C) and nitrogen (δ15N) isotope signatures of toenails of dunlins captured during winter at reference sites along the East-Atlantic Flyway, from Mauritania to the United Kingdom. Afterwards, during spring migration, dunlins were sampled at the Tagus estuary, Portugal, and assigned to their wintering grounds according to their stable isotope signatures. Toenails from wintering dunlins at different sites had significantly different δ13C and δ15N signatures, despite some overlap in isotopic carbon ratios of birds from Morocco, Portugal and the UK. Among birds sampled during migration in Portugal, we found a clear bimodal pattern in δ13C values, corresponding to passage migrants from Mauritania (enriched δ13C values) and wintering birds from the Tagus estuary (depleted δ13C values). The first passage migrants from Mauritania appeared at the Tagus estuary by the end of March, with peak numbers during late April and early May. Our study provides evidence that isotopic signatures of toenails can play a determinant role in tracing the wintering origins of migrant dunlins at their stopover areas. Toenails, instead of feathers, can be the powerful and innovating tissue to sample in wader studies, allowing to bridge the gap in the field of migratory connectivity between sites used in different phases of the life cycle of waders.