The thick-shelled river mussel Unio crassus is critically endangered throughout its range as a result of increasing human activity and habitat loss. Next generation DNA sequencing was used to develop a set of microsatellite markers that can be used for future ecological and population genetics studies of U. crassus. A total of 11 polymorphic loci were identified and characterized using 57 individuals from two Polish populations. Numbers of alleles ranged from 2 to 15 and expected heterozygosity levels ranged from 0.069 to 0.899. Deficiency of heterozygous genotypes was observed in four loci. Marker independence was confirmed with tests for linkage disequilibrium, however, analyses indicated evidence of null alleles in four loci. The microsatellite markers developed specifically for U. crassus provide a valuable tool for future ecological, population genetic assessments, and conservation management of this species. J. Exp. Zool. 319A:113–116, 2013. © 2013 Wiley Periodicals, Inc.