The Atlantic Iberian brown trout is at the southwestern limit of its distribution. At this ecological edge, which was once a glacial refugia, anadromy becomes less common as increased water temperatures restricted populations closer to the headwaters. We examined 847 individuals from 20 populations from throughout this region and assessed spatial genetic structure using 11 protein and four microsatellite loci. The higher levels of heterozygosity and allelic diversity north of the southernmost limit of anadromy (SLA), as well as an isolation-by-distance model of population structure, likely influenced by the anadromous forms, suggest that more stable demographic conditions existed over time in this region. Populations south of the SLA were highly differentiated given the restricted size of the area (protein F(ST) = 0.16 in the north and 0.63 in the south of the SLA; microsatellite F(ST) = 0.18 in the north and 0.70 in the south of the SLA). The low levels of heterozygosity and the pattern of southward allele depletion in resident populations is indicative of fragmentation, caused by stressful ecological conditions that reduced the anadromy (restricting gene flow) and the effective population sizes (higher genetic drift), which, in combination, dramatically decreased within-population genetic variation and increased among-population genetic variation. The higher gene diversity north of the SLA does not reflect ancestry but rather the signature of a population size expansion, as evidence suggest the persistence of older populations (with several private alleles) south of the SLA. These data support a scenario that demonstrates how contemporary events (critical ecological conditions) can moderate historical influences, suggesting that careful interpretation of the evolutionary history of glacial refugia is necessary, especially where populations persisted for a long time but not always with optimal ecological conditions. These peripheral populations are of high conservation value and should be preserved to help conserve the future potential of the species.