Several Diaporthe species are recognized as causal agents of many plant disease symptoms, including twig and branch cankers, dieback, shoot blight, and root and fruit rots. In Uruguay, the proximity between apple, pear and peach orchards offers the possibility to study the presence of different Diaporthe spp. associated with wood cankers across different deciduous fruit trees. Symptomatic twigs and branches of these orchard species were sampled, and isolates of Diaporthe were obtained. Selected isolates were used for cross inoculations in the three hosts. Seven Diaporthe spp. were identified, based on the internal transcribed spacer (ITS) region of rDNA and the translation elongation factor 1-alpha gene (EF1-a) phylogenies. The species were: Diaporthe amygdali, D. foeniculina, D. infecunda, D. eres, D. terebinthifolii, D. oxe and D. phaseolorum, while two isolates Diaporthe sp. 1 and Diaporthe sp. 4 could not be assigned to any species. Diaporthe infecunda, D. eres, D. terebinthifolii, D. phaseolorum and D. oxe on Pyrus communis and D. foeniculina on Malus domestica represent new records in these hosts in Uruguay, while D. oxe isolated from Prunus persica is a new record for this species. Diaporthe eres and D. phaseolorum were the most virulent species, posing the greatest risk due to their wide distribution and virulence in apple and peach trees. Although pear trees showed less symptomatic tissues and were less susceptible than peach and apple trees in the pathogenicity tests, they harboured seven of the species, and therefore should be considered as reservoirs of Diaporthe in Uruguayan orchards. Trees of the three hosts could be considered potential reciprocal sources of pathogenic Diaporthe spp.