Abstract: We investigated the genetic structure of 19 colonies of the myrmeciine ant Myrmecia pyriformis Smith, 1858, using five microsatellite loci. Nestmate relatedness of b = 0.51  0.04 significantly deviated from the theoretical value of 0.75, which is expected in a monogynous monandrous social system. The occurrence of multiple mating by queens was the most likely explanation in the majority of colonies, and the effective number of male mates was estimated to be 2.60. However, multiple female reproductives were also inferred in some cases. The results correspond well both to observations on promiscuous mating behaviour in the winged sexuals of some species of Myrmecia and to the occurrence of gamergates (mated workers) in M. pyriformis. The finding of high levels of polyandry in a species with totipotent workers is discussed with regard to the evolution of eusociality.