Abstract The genetic identity and conservation status of lake ecotype of brown trout (Salmo trutta) populations in Slovenia’s Alpine lakes Bled and Bohinj have been subjected to changes due to historical stocking practices. We aimed to evaluate the genetic differentiation and origins of these populations to advise for correct conservation strategies. Using microsatellite and mitochondrial DNA analysis on 365 trout specimens, we explored the genetic diversity and structure of these populations, comparing them with potential originators. Results revealed distinct genetic profiles between Bled and Bohinj lake trout, with Bohinj lake trout closely aligned with the native Danubian lineage and Bled lake trout showing a hybrid composition, significantly influenced by hatchery-introduced Atlantic lineage strains. The genetic distinction between these populations underscores the complexity of their conservation, showcasing the impact of human-mediated introductions on natural populations. The urgent need for conservation measures to preserve the Bohinj population’s native genetic pool is highlighted, underlining the must to prevent the transfer of individuals from Lake Bled or elsewhere and to reconsider stocking practises in order to prevent further genetic dilution of these iconic European lake dwellers.