Pollution is one of the main concerns in marine ecosystems nowadays. Environmental anthropogenic-mediated toxicants may affect genetic diversity both at the individual and ecosystem levels and may also alter the genetic structure of populations. This study examined the temporal pattern of genetic diversity among populations of the benthic bivalve Ensis siliqua in two locations of Galicia, following the Prestige oil spillage. On November 13, 2002 the oil tanker Prestige sank at 240 km from Galician coast and 63,000 tonnes of heavy fuel were released to the marine environment. E. siliqua samples were sampled between 2001 and 2006. Genetic variation was assessed by means of Random Amplification of Polymorphic DNA (RAPD). A significant decrease in genetic diversity was observed for the 2006 samples. Nei's genetic distance, fixation index (PhiPT), and PCA values also supported differences in the 2006 samples. We hypothesize that the temporal genetic variation observed in E. siliqua populations is due to a strong effect of genetic drift caused by a reduction in population size and that the indirect effects of the Prestige spill possibly caused this reduction.