Grosmannia alacris is a fungus commonly associated with root-infesting bark beetles occurring on Pinus spp. The fungus has been recorded in South Africa, the USA, France, Portugal and Spain and importantly, has been associated with pine root diseases in South Africa and the USA. Nothing is known regarding the population genetics or origin of G. alacris, although its association with root-infesting beetles native to Europe suggests that it is an invasive alien in South Africa. In this study, microsatellite markers together with newly developed mating type markers were used to characterize a total of 170 isolates of G. alacris from South Africa and the USA. The results showed that the genotypic diversity of the South African population of G. alacris was very high when compared to the USA populations. Two mating types were also present in South African isolates and the MAT1-1/MAT1-2 ratio did not differ from 1:1 (χ2 = 1·39, P = 0·24). This suggests that sexual reproduction most probably occurs in the fungus in South Africa, although a sexual state has never been seen in nature. In contrast, the large collection of USA isolates harboured only a single mating type. The results suggest that multiple introductions, followed by random mating, have influenced the population structure in South Africa. In contrast, limited introductions of probably a single mating type (MAT1-2) may best explain the clonality of USA populations.