Background and Aims The frequency at which males can be maintained with hermaphrodites in androdioecious populations is predicted to depend on the selfing rate, because self-fertilization by hermaphrodites reduces prospective siring opportunities for males. In particular, high selfing rates by hermaphrodites are expected to exclude males from a population. Here, the first estimates are provided of the mating system from two wild hexaploid populations of the androdioecious European wind-pollinated plant M. annua with contrasting male frequencies.Methods Four diploid microsatellite loci were used to genotype 19-20 progeny arrays from two populations of M. annua, one with males and one without. Mating-system parameters were estimated using the program MLTR.Key Results Both populations had similar, intermediate outcrossing rates (t(m) = 0.64 and 0.52 for the population with and without males, respectively). The population without males showed a lower level of correlated paternity and biparental inbreeding and higher allelic richness and gene diversity than the population with males.Conclusions The results demonstrate the utility of new diploid microsatellite loci for mating system analysis in a hexaploid plant. It would appear that androdioecious M. annua has a mixed-mating system in the wild, an uncommon finding for wind-pollinated species. This study sets a foundation for future research to assess the relative importance of the sexual system, plant-density variation and stochastic processes for the regulation of male frequencies in M. annua over space and time.