Queen polymorphism in wing morphology and thoracic structure provides the opportunity to test hypotheses about mating strategies and colony founding modes. Some studies indicate that the difference in mating behavior between winged and wingless queens may promote genetic isolation, possibly leading to speciation. However, the knowledge about genetic differences and phylogenetic relationships among polymorphic queens is limited. Queens of the myrmicine ant Vollenhovia emeryi Wheeler exhibit two morphs: a long-winged (L-queen) and a short-winged (S-queen) morph. We analyzed the phylogenetic relationship among populations of L- and S-queens in V. emeryi and the congeneric species V. nipponica, V. benzai, V. okinawana, and V. yambaru. The molecular phylogeny inferred from mtDNA (≈2,200 nt) showed that S-queens formed a monophyletic clade and that L- and S- queens sampled from the same location did not group together. The phylogeny indicates that wing reduction occurred only once and that S-queen populations are genetically differentiated from L-queen populations, at least in their maternal genomes. The phylogeny is consistent with the hypothesis that wing reduction leads to reproductive isolation in V. emeryi.