The Eurasian Black Tern, Chlidonias niger niger, nests in marshes continuously distributed across Eurasia, but migration routes, staging sites, and non-breeding distributions are not well understood. In western Europe some populations have declined substantially over several decades (>90%), thus a more complete understanding of breeding site connectivity and migratory routes is needed. We collected tissue samples of terns in breeding colonies in the Netherlands, Latvia, southern Ukraine, eastern Siberian Russia, and from individuals at one important post-nuptial staging site in The Netherlands. Microsatellite data suggest significant differentiation among all breeding sites and the pattern is supported by differences among sites in most morphological measures. Conversely, mitochondrial DNA suggests similarity and population expansion especially from the region around Ukraine. We assigned 70% of the birds sampled on the staging site to the Netherlands/Latvia breeding population, but none to the southern Ukrainian or eastern Russian population. Our data indicated limited contact at post-nuptial staging sites contribute to genetic structure among breeding sites for this species. Our study demonstrates the utility of genetic data in migration studies to delineate migratory flyways and highlight the importance of specific staging sites to specific breeding subpopulations.