The North American spiny-cheek crayfish, Orconectes limosus (Cambaridae), endangered in its native range, is a widespread invasive species in European waters and conservationally important carrier of crayfish plague. However, its population structure is poorly known, and no informative genetic markers for the species are available. We tested cross-species transfer of microsatellite loci to spiny-cheek crayfish from 5 other crayfish species. Variability of 10 successfully amplifying loci derived from 4 species was then tested in 60 individuals of O. limosus originating from 3 natural populations: the river Danube at Bogyiszló in Hungary, a pond in Starý Klíèov, and the brook Eernovický, both in the Czech Republic. The allele number within the populations ranged from 4 to 10 alleles per locus, while heterozygosity levels varied from 0.650 to 0.900 for H(o) and from 0.660 to 0.890 for H(e). No linkage disequilibrium and no null alleles were detected. The selected markers are useful for assessing population structure, intraspecific variation, and paternity studies in spiny-cheek crayfish.