In colonies of the queen-polymorphic ant Vollenhovia emeryi, some colonies produce only long-winged (L) queens, while others produce only short-winged (S) queens. At four nuclear microsatellite loci, males in the S colony had alleles that were different from those of their queen. This suggests that the nuclear genome of males is not inherited from their colony queen, as has also been described for Wasmannia auropunctata (Roger). In V emeryi the possibility of male transfer from other colonies has not been ruled out because previous studies of this species have obtained only nuclear gene information. We analyzed both mitochondrial and nuclear genes for S queens, S males and L queens to clarify the origins of males. Sequence analyses showed that although S queens and S males shared the same mtDNA haplotype, they had a different genotype at a nuclear gene (long-wavelength opsin) locus. Neighbor-joining analysis based on the four microsatellite loci also suggested gene pool separation between S queens and S males. These results are consistent with predictions of clonal reproduction by males. While L queens share opsin genotypes with S males, they have very different mtDNA sequences. Hybridization in the near past between S queens and L males or gene transmission from S males to L queen populations in the present would explain these differences.