Teratosphaeria destructans is an aggressive fungal pathogen causing leaf and shoot blight on young Eucalyptus trees in plantations. The disease occurs across tropical and subtropical regions of South East Asia and has recently been found in South Africa. Asexual structures of the pathogen are produced on infected tissues, but sexual structures have never been observed. The aim of this study was to investigate the reproductive biology of T. destructans by characterising its mating type (MAT1) locus and investigating its potential for sexual recombination. We found that T. destructans has a heterothallic mating system, with either the MAT1-1-1 and MAT1-1-10 genes (MAT1-1 idiomorph) or the MAT1-2-1 and MAT1-2-12 genes (MAT1-2 idiomorph) present in a single individual. With a multiplex PCR assay, it was possible to distinguish the two MAT idiomorphs in several Teratosphaeria species and this approach was applied to six global populations of T. destructans. Although both mating types occurred in the South East Asian populations, a single mating type dominated each population. Isolates from the recent disease outbreak in South Africa comprised only a single mating type. Attempts to induce a sexual cycle in vitro using strains of opposite mating type were not successful. The uneven distribution of mating types in populations of T. destructans and the presence of only an asexual state on infected tissues suggests the absence of or at least a minor role for sexual reproduction where the pathogen occurs on non-native Eucalyptus in plantations.