Thousands of microsatellite loci were identified in the nemertean Prosorhochmus americanus through enrichment-isolation and next-generation sequencing protocols. We tested 48 loci for amplification and subsequently genotyped 17 loci in 30 individuals from three North American populations. Most loci were polymorphic within and across populations; six were monomorphic. The number of alleles per locus ranged from 1 to 9 (mean 4.35, SD 2.37). Cross-species amplification in four nemertean species revealed extended marker utility. These loci will enable investigations of colonization potential and natural history in this selfing, putatively introduced species.