Genetic variation was assayed at 14 allozyme loci in estuarine, coastal and offshore samples of Lozano's goby, Pomatoschistus lozanoi and the sand goby, P. minutus . Samples were taken from locations on the Belgian Continental Shelf and in the Schelde estuary with a range of environmental heterogeneity. We evaluate whether any differences in (1) the degree of genetic variation and (2) allele frequencies at the various loci exist within samples occurring in various habitats on the BCS and in the Schelde estuary. No significant differences in levels of genetic diversity were recorded between estuarine, coastal and offshore samples in either species. A temporally stable clinal gradient in allele frequencies at the two-allele locus GPI-A* was observed in P. lozanoi , differentiating the samples in an estuarine, coastal and offshore group. We suggest that these differences might be maintained by balancing selection at locus GPI-A*.