Conservation of species should be based on knowledge of effective population sizes and understanding of how breeding tactics and selection of recruitment habitats lead to genetic structuring. In the stream-spawning and genetically diverse brown trout, spawning and rearing areas may be restricted source habitats. Spatio–temporal genetic variability patterns were studied in brown trout occupying three lakes characterized by restricted stream habitat but high recruitment levels. This suggested non-typical lake-spawning, potentially representing additional spatio–temporal genetic variation in continuous habitats. Three years of sampling documented presence of young-of-the-year cohorts in littoral lake areas with groundwater inflow, confirming lake-spawning trout in all three lakes. Nine microsatellite markers assayed across 901 young-of-the-year individuals indicated overall substantial genetic differentiation in space and time. Nested gene diversity analyses revealed highly significant (≤P = 0.002) differentiation on all hierarchical levels, represented by regional lakes (FLT = 0.281), stream vs. lake habitat within regional lakes (FHL = 0.045), sample site within habitats (FSH = 0.010), and cohorts within sample sites (FCS = 0.016). Genetic structuring was, however, different among lakes. It was more pronounced in a natural lake, which exhibited temporally stable structuring both between two lake-spawning populations and between lake- and stream spawners. Hence, it is demonstrated that lake-spawning brown trout form genetically distinct populations and may significantly contribute to genetic diversity. In another lake, differentiation was substantial between stream- and lake-spawning populations but not within habitat. In the third lake, there was less apparent spatial or temporal genetic structuring. Calculation of effective population sizes suggested small spawning populations in general, both within streams and lakes, and indicates that the presence of lake-spawning populations tended to reduce genetic drift in the total (meta-) population of the lake.