27Currently, eight Phyllosticta species are known to be associated with Citrus hosts, 28 incorporating endophytic and pathogenic lifestyles. As sexual reproduction is a key factor 29 involved in host-interaction, it could be related to the differences in lifestyle. To evaluate this 30 hypothesis, we characterized the mating-type loci of six Citrus-associated Phyllosticta species 31 from whole genome assemblies. Mating-type genes are highly variable in their sequence 32 content, but the genomic locations and organization of the mating-type loci are conserved. 33Phyllosticta citriasiana, P. citribraziliensis and P. paracitricarpa are heterothallic, and P. 34 citrichinaensis was confirmed to be homothallic. In addition, the P. citrichinaensis MAT1-2 35 idiomorph occurs in a separate location from the mating-type locus. Ancestral state 36 reconstruction suggests that homothallism is the ancestral thallism state in Phyllosticta, with a 37 shift to heterothallism in Phyllosticta species that are pathogenic to Citrus. Moreover, the 38 homothallic strategies of P. capitalensis and P. citrichinaensis result from independent 39 evolutionary events. As the pathogenic species P. citriasiana, P. citricarpa and P. 40 paracitricarpa are heterothallic and incapable of selfing, disease management practices 41 focused in preventing the occurrence of sexual reproduction could assist in the control of 42Citrus Black Spot and Citrus Tan Spot diseases. This study emphasizes the importance of 43 studying Citrus-Phyllosticta interactions under evolutionary and genomic perspectives, as 44 these approaches can provide valuable information about the association between Phyllosticta 45 species and their hosts, and also serve as guidance for the improvement of disease 46 management practices. 47 48