Understanding the links between breeding and wintering areas of migratory species has important ecological and conservation implications. Recently, stable isotope technology has been used to further our understanding. Stable isotope ratios vary geographically with a range of biogeochemical factors and isotope profiles in organisms reflect those in their food and environment. For inert tissues like feathers, isotope profiles reflect the environment in which they were formed. Following large-scale habitat destruction, the globally threatened aquatic warbler Acrocephalus paludicola has a fragmented breeding population across central Europe, largely in Belarus, Poland and Ukraine. The species' sub-Saharan African wintering grounds have not yet been discovered, and this significantly hampers conservation efforts. Aquatic warblers grow their flight feathers on their wintering grounds, and we analysed stable isotope ratios (delta(15)N, delta(13)C, delta D) in rectrices of adults from six main breeding sites (subpopulations) across Europe to determine whether different breeding subpopulations formed a single mixed population on the wintering grounds. delta(15)N varies considerably with dietary trophic level and environmental factors, and delta D with the delta D in rainfall; neither varied between aquatic warbler subpopulations. Uniform feather delta(15)N signatures suggest no major variation in dietary trophic level during feather formation. High variance and inter-annual differences in mean delta D values hinder interpretation of these data. Significant differences in mean delta(13)C ratios existed between subpopulations. We discuss possible interpretations of this result, and consider differences in moulting latitude of different subpopulations to be the most parsimonious. delta(13)C in plants and animals decreases with latitude, along a steep gradient in sub-Saharan Africa. Birds from the most northwesterly breeding subpopulation (Karsibor, Poland) had significantly lower variance in delta(13)C and delta(15)N than birds from all other sites, suggesting either that birds from Karsibor are less geographically dispersed during moult, or moult in an area with less isotopic heterogeneity. Mean delta(13)C signatures from winter-grown feathers of different subpopulations were positively correlated with the latitude and longitude of breeding sites, suggesting a strong relationship between European breeding and African winter moulting latitudes. The use of stable isotopes provides novel insights into migratory connectivity and migration patterns in this little-known threatened species.