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We examined the long‐term temporal (1910s to 1990s) genetic variation at eight microsatellite DNA loci in brown trout (Salmo trutta L) collected from five anadromous populations in Denmark to assess the long‐term stability of genetic composition and to estimate effective population sizes (N_e). Contemporary and historical samples consisted of tissue and archived scales, respectively. Pairwise θ_ST estimates, a hierarchical analysis of molecular variance (amova) and multidimensional scaling analysis of pairwise genetic distances between samples revealed much closer genetic relationships among temporal samples from the same populations than among samples from different populations. Estimates of N_e, using a likelihood‐based implementation of the temporal method, revealed N_e ≥ 500 in two of three populations for which we have historical data. A third population in a small (3 km) river showed N_e ≥ 300 …