The Eucalyptus stem canker pathogens Teratosphaeria gauchensis and T. zuluensis (Capnodiales, Teratosphaeriaceae) are found in many tropical regions of the world where their hosts are cultivated for plantation forestry. Population genetic analyses have suggested that some populations undergo recombination, even though their sexual states have never been observed. Against this background, the aim of this study was to characterize the mating type (MAT) locus of these species and thus to better understand the basis of their diversity. Known Mycosphaerellaceae MAT genes were used to identify and investigate the MAT locus in the T. gauchensis and T. zuluensis genomes. Both species were found to be heterothallic and primers were designed to amplify the opposite MAT idiomorphs as well as conserved regions within the MAT1‐1‐1 and MAT1‐2‐1 genes. Each Teratosphaeria MAT idiomorph was defined by either the MAT1‐1‐1 or the MAT1‐2‐1 gene, and an idiomorph‐specific hypothetical protein (MAT1‐1‐10 and MAT1‐2‐12). Populations of T. zuluensis from Asia and southern Africa were dominated by a single mating type, whereas the proportions of the different idiomorphs for T. gauchensis in South America and southern Africa were similar. There was no physical evidence of sexual reproduction for either species and it is argued that although recombination may be possible, it is unlikely to form an important part of their life cycles in diseased Eucalyptus plantations. Instead, continuous human‐mediated multiple introductions of these species have probably resulted in the current genetic structure of their populations, which holds risk for future disease outbreaks and interspecific hybridization.