The family Rosaceae includes a large number of species ranging from herbaceous (Fragaria) to ornamental plants (Rosa and Pyracantha) and fruit trees (Malus and Pyrus). Diaporthe species have been associated with twig canker, shoot blight, dieback, wood decay and fruit rot on members of the Rosaceae. In this study a collection of isolates from several Rosaceae hosts were characterised by multi-locus sequence analyses using the internal transcribed spacer, translation elongation factor 1-alpha, beta-tubulin, histone H3 and calmodulin loci. The phylogenetic analyses of the combined five loci revealed that the isolates studied were distributed among four clades, of which two correspond to D. foeniculina and D. eres. The other two clades, closely related to D. passiflorae and D. leucospermi represent two new species, D. pyracanthae sp. nov. and D. malorum sp. nov., respectively. Further, pathogenicity assays have shown that of the four species tested, D. malorum was the most aggressive species on apple fruit and D. eres was the most aggressive species on detached pear twigs. A revision of all Diaporthe (and Phomopsis) names that have been associated with Rosaceae hosts as well as their current status as pathogens of members of this family is presented.