Population substructure of the cuttlefish Sepia officinalis (Mollusca: Cephalopoda), as investigated by genetic variation of microsatellite loci, has been reported to be significantly extensive around the Iberian Peninsula with FST = 0.061 (Pérez-Losada et al. 2002) and panmictic in the semi-enclosed Adriatic Sea with FST = 0.011 (Garoia et al. 2004). Yet, no verified genetic information on population substructure existed for the northern distribution range of this species in the English Channel and Bay of Biscay.So far, reproductive and migration behaviour and in-vitro oxygen binding properties of haemocyanin have suggested existence of separate populations in the English Channel and the Bay of Biscay. Examination of genetic variation at seven microsatellite loci within samples from the Bay of Biscay, the English Channel and the southern North Sea indicated low levels of genetic differentiation in this area but also a breakdown of free gene flow at highly significant average FST = 0.018. Although there is a considerable genetic exchange between populations of S. officinalis in the English Channel and the Bay of Biscay, they cannot be regarded as a single, freely interbreeding population. Earlier reported biological differences might thus be due to genetic variability between the populations.