In ants of the genus Myrmica, female progeny may be the offspring of one to several different queens. In addition, both workers and queens are capable of producing haploid male offspring. Even in such complex colonies, parentage can be assigned on the basis of genotypic variation at highly polymorphic simple sequence repeat loci. Methods are described for isolating and screening dinucleotide repeat loci in ants. Three independent loci, Myrt2, Myrt3 and Myrt4, show expected heterozygosities of 0.94, 0.92 and 0.95, respectively. When used in parallel these loci should be sufficient to establish parentage in the vast majority of screened colonies. An initial screening indicates that males are produced by workers in the subalpine ant Myrmica ‘near tahoensis’.