European flounder (Platichthys flesus) and plaice (Pleuronectes platessa) are commercially important marine fish species inhabiting the continental shelf waters of Europe. Morphological similarity between the two makes it difficult to identify their hybrids, so species misclassification can generate errors in defining stocks in terms of their conservation and management. Flounder and plaice populations from the North Sea and the Baltic Sea were studied. Mitochondrial DNA (mtDNA) was used to confirm the morphological species identification. The set of molecular markers, two mitochondrial (cytochrome b and D-loop) and two nuclear (the ribosomal marker ITS and parathyroid hormone-related protein gene), was constructed to identify the two flatfish species and their hybrids. “Pure” flounder (P. flesus) were observed in the Bay of Gdańsk, Baltic Sea, and off the coast of Denmark in the North Sea. The fishing area near Bornholm in the Baltic is rich in P. flesus × P. platessa hybrids. The length difference of the amplified D-loop fragment was used for species identification. The characteristics of heteroplasmy in the control region (D-loop) can be useful as a population marker in the European flounder. Our studies demonstrate the utility of mtDNA polymorphism combined with nuclear molecular markers for correct identification of the morphologically similar and hybridized European flounder and plaice.