The European plaice (Pleuronectes platessa; Pleuronectidae) is considered the most important flatfish for fisheries in Europe. Overall catches have been reduced drastically throughout the past decades, indicating a severe decline in the natural populations. However, a limited set of genetic tools are available to improve stock assessment and management for the species. Sequences of European plaice and its congener, the winter flounder (Pleuronectes americanus) deposited in the public database were searched for short tandem repeats. Out of 22 loci derived from database sequences, eight were amplified successfully and genotyping of 23 wild-caught individuals showed expected heterozygosities ranging from 0.18 to 0.84. The average allele number was 4.75 per locus (range 2–11). Successful cross-species amplification of the markers was demonstrated from two related flatfishes. All eight markers were successfully amplified from the European flounder (Platichthys flesus), a phylogenetically closely related species, whereas three of them worked in Achiroides melanorhynchus, an endemic species of Southeast Asia for which no microsatellites have been described to date.