Ostrea chilensis (Küster, 1844), the flat oyster, is native to Chile and New Zealand. In Chile, it occurs in a few natural beds, from the northern part of Chiloé Island (41 ºS) to the Guaitecas Archipelago (45 ºS). This bivalve is slow growing, broods its young, and has very limited dispersal potential. The Ostrea chilensis fishery has been over-exploited for a number of decades such that in some locations oysters no longer exist. The aim of this study was to study the genetic diversity of the Chilean flat oyster along its natural distribution to quantify the possible impact of the dredge fishery on wild populations. The genetic structure and diversity of Ostrea chilensis from six natural beds with different histories of fishing activity were estimated. Based on mitochondrial (Cytb) and nuclear (ITS1) DNA sequence variation, our results provide evidence that genetic diversity is different among populations with recent history of wild dredge fishery efforts. We discuss the possible causes of these results. Ultimately, such new information may be used to develop and apply new management measures to promote the sustainable use of this valuable marine resource.