Chrysoporthe syzygiicolaandC. zambiensisare ascomycete tree pathogens first described from Zambia, causing stem canker onSyzygium guineenseandEucalyptus grandis, respectively. The taxonomic descriptions of these two species were based on their anamorphic states, as no sexual states are known. The main purpose of this work was to use whole genome sequences to identify and define the mating-type (MAT1) loci of these two species. The uniqueMAT1loci forC. zambiensisandC. syzygiicolaconsist of theMAT1-1-1,MAT1-1-2, andMAT1-2-1genes, but theMAT1-1-3gene is absent. Genes canonically associated with opposite mating types were present at the single mating-type locus, suggesting thatC. zambiensisandC. syzygiicolahave homothallic mating systems.