Contrasting the general trend of declining Afro-Palearctic migratory bird populations, the Whiskered Tern (Chlidonias hybrida hybrida) has recently exhibited spectacular demographic expansion and range shifts across its patchy European breeding distribution. To explore population connectivity of this species, individuals sampled from four sites in France, Poland, and Ukraine (n = 78) were genotyped at six microsatellite DNA loci and one partial mitochondrial cytochrome-b gene. Genetic variation was generally high (H_E = 0.47-0.67; π = 1.29-2.26). Contrasting the historical absence of significant genetic structure (Φ_ST = 0.061-0.027), the microsatellite data indicated strong differentiation between France and the Eastern European sites (F_ST = 0.067-0.098). This, coupled with evidence of two distinct genetic clusters (K = 2; ΔK = 2) corresponding to little subdivision between Poland and Ukraine, has confirmed the presence of two European Whiskered Tern subpopulations (western and eastern) characterized by stark differences in wintering distribution and seasonal migratory routes.