Cellulose acetate electrophoresis of allozymes was used to differentiate the two endangered Mediterranean killifishes,Aphanius iberusandA. fasciatus.Thirty‐eight putative enzyme‐coding loci were examined in two populations from the Albuixec lagoon (Spain) and from the Biguglia coastal pond (Corsica). Low within‐population polymorphism was detected. The mean number of al‐leles per locus, percentage of polymorphic loci, and heterozygos‐ity were A = 1.2 (SE 0.1), P99= 21.1%, H = 0.050 (SE 0.018) inA. iberus,and A = 1.3 (SE 0.1), P99= 28.9%, H = 0.057 (SE 0.019) inA. fasciatus.A general concordance with the Hardy‐Weinberg equilibrium was detected. Allelic distribution for 13 loci proved diagnostic at species level. The genetic distance between the two species analysed was relatively high (D = 0.539), indicating considerable genetic divergence between the two congeneric species. These results represent a valuable basis for further studies on the genetic structure of Mediterranean cyprinodontids to ensure sustainable management of these species.