The flat oyster (Ostrea edulis) is one of the most appreciated molluscs in Europe, but natural beds have been greatly reduced due to harvesting and the effects of the parasite Bonamia ostreae. Characterization of current wild populations is required to develop long-term bed restoration programmes by enhancing genetic diversity and tolerance to bonamiosis. Oysters from different locations corresponding to the main natural beds from Denmark, The Netherlands, England, Ireland, France and Spain, including two different cohorts per location were sampled in 2011 and 2013. Sixteen microsatellite loci were used to study temporal and geographical genetic structure. Temporal variation was low, although sometimes significant probably due to high variance in reproductive success among individuals. Conversely, samples from different countries showed much higher genetic divergence, and Ireland and France presented differences among locations within country. Clustering analyses grouped samples into three main geographical regions, associated with oceanic fronts: one group constituted by The Netherlands and Denmark; another by France, Ireland and England; and a third one exclusively by Spain. Effective population sizes (Ne) within regions were high (>1000), which reflects population stability and low levels of genetic drift. The presence of regional genetic structure shows the potential for local adaptation of O. edulis and suggests caution when transplanting individuals, especially between distant geographical regions.