AbstractMediterranean populations of brown trout (Salmo trutta L. complex) have lost a large part of their genetic distinctiveness, mostly due to massive restocking, and the waters of the Gardens of Ninfa (province of Latina, central Italy, Site of Community Importance since 2013) are regarded as one of a few potential reservoirs of autochthonous trout lineages in the Tyrrhenian drainage of the Italian peninsula. In this study, nuclear and mitochondrial markers were used on brown trout samples from Ninfa to estimate non‐Mediterranean influence in the population gene pool, potential changes of genetic structure over time and genetic relationships with other sites known (or suspected) to host native trout gene pools. Striking changes in both microsatellite and mtDNA allele frequencies over a 9‐year time span were found and provided evidence of unrecorded stocking from the nearby Lake Fibreno. Results are analysed in the light of potential ecological consequences of such events on a longer time scale and provide a scientific background for fisheries management and conservation programmes in the area.