Since the 1980s, heavy exploitation has led to severe population declines in winter flounder, Pseudopleuronectes americanus, a demersal flatfish inhabiting the inshore bays and estuaries of the Northwest Atlantic. Recent studies using a limited number of non-specific microsatellite loci have demonstrated fine-scale population structure. Here, we present 21 novel species-specific microsatellite loci to enhance studies of genetic diversity, population differentiation, effective population size and mating behavior of this important commercial species.