Mercurialis annua is a wind-pollinated annual showing a remarkable sexual-system variation, with hexaploid populations being either monoecious or androdioecious. Hexaploid M. annua is most likely a product of hybridization between diploid M. huetii and tetraploid M. annua; therefore, we developed microsatellite loci by isolating simple sequence repeat (SSR) sequences from the diploid progenitor, cross-amplification tests in M. huetii/M. annua species complex followed by selection of loci amplifying only in M. huetii and hexaploid M. annua, and testing polymorphism in 1 hexaploid population. This protocol resulted in 10 unlinked, polymorphic loci amplifying 4-10 alleles per locus. Due to specific amplification of the diploid part of the genome originating from M. huetii, these loci produce codominantly scored, diploid data for allohexaploid species, thereby simplifying data collection and subsequent analyses. Sequencing of the hexaploid polymerase chain reaction product for all 10 loci and aligning it with M. huetii SSR library sequence confirmed orthology of the characterized loci. Inheritance tests in 4 hexaploid crosses confirmed diploid Mendelian segregation of the new loci.