Fungi of Guignardia genus are commonly isolated from different plant species and most of the time, they are characterized as endophytes. However, some species of this genus, like G. citricarpa and G. psidii are known as causal agents of serious diseases that affect important crops such as Citrus Black Spot and guava fruit rot, respectively. They are also responsible for diseases that cause foliar spots in different fruit species and also in other crops, but cause minor damages. Despite evidences that G. mangiferae colonizes different plant species, there are few studies about its genetic diversity associated with different hosts. This work has the objective to characterize Guignardia isolates obtained from different hosts and tissues by RAPD, fAFLP and DNA sequence of ITS1-5.8S-ITS2 region, as well as to develop pathogenicity tests through cross inoculation in citrus and guava fruits. It was observed that molecular markers were able to discriminate isolates of different Guignardia species. Pathogenicity tests showed that G. citricarpa caused CBS symptoms on citrus fruits, but it did not produce any symptoms in guava fruits. G. mangiferae isolates were able to cause rot symptoms on guava fruits, but they have not produced any symptoms on citrus fruits. Guignardia isolates obtained from mango leaves that have not been classified in species have not presented any symptoms in citrus and guava fruits. Although G. mangiferae is commonly isolated asymptomatically in different plants, this work supports the evidence that this species has a latent pathogen behavior, at least for guava plants.