The intense fishing mortality imposed on Atlantic cod in Icelandic waters during recent decades has resulted in marked changes in stock abundance, as well as in age and size composition. Using a molecular marker known to be under selection (Pan I) along with a suite of six neutral microsatellite loci, we analysed an archived data set and revealed evidence of distinct temporal changes in the frequencies of genotypes at the Pan I locus among spawning Icelandic cod, collected between 1948 and 2002, a period characterized by high fishing pressure. Concurrently, temporal stability in the composition of the microsatellite loci was established within the same data set. The frequency of the Pan IBB genotype decreased over a period of six decades, concomitant with considerable spatial and technical changes in fishing effort that resulted in the disappearance of older individuals from the fishable stock. Consequently, these changes have likely led to a change in the genotype frequencies at this locus in the spawning stock of Icelandic cod. The study highlights the value of molecular genetic approaches that combine functional and neutral markers examined in the same set of individuals for investigations of the selective effects of harvesting and reiterates the need for an evolutionary dimension to fisheries management.