Overlapping external morphometric characters easily confound the flatfishes Solea aegyptiaca and Solea solea (Soleidae) in areas of the Mediterranean Sea where both species live in sympatry. This leads to uncertainties in the fisheries and marketing of the species, in addition to misinterpretations in biogeography and conservation studies. Here we describe a simple restriction fragment length-based diagnostic test, which differentiates S. solea from S. aegyptiaca, as well as from other species of the Soleidae family. Furthermore, we show that the two species living in sympatry in the Gulf of Kavala (North Aegean Sea, Greece) present significant qualitative differences in muscle fatty acid composition, a property which can also be used to distinguish the two cryptic species.