Prospects for estimation of parameters of models of sperm competition from field data have improved recently with the development of methods that employ multilocus genotype data from brood-structured samples. Sperm competition in Drosophila buzzatii is of special interest because it is possible to directly observe the breeding behaviour of this species in its natural habitat of rotting cactus. Previous laboratory experiments showed that this species exhibits an unusual pattern of frequent remating and sperm partitioning. This paper reports the first attempt to estimate the frequency of female remating and sperm competition in natural populations of D. buzzatii. For the Australian population studied, the mean remating frequency was lower (α = 2.12–2.20) than previously estimated in laboratory experiments with the same population, whereas mean sperm displacement (β = 0.69–0.71) fell within the limits of previous laboratory results. The evolution of the D. buzzatii mating system is discussed.