The ascomycete genus Diaporthe includes plant pathogens and endophytes on a wide range of hosts including economically important crops. Anamorphs are coelomycetous and reside in the genus Phomopsis. Phomopsis amygdali is the causal agent of twig canker and blight of almonds. In a recent survey of dieback of almonds in Portugal, the most frequent fungi detected were Diaporthe/Phomopsis species. Isolates from almond and other Prunus species were characterised and grouped according to their microsatellite-primed PCR (MSP-PCR) profiles and representatives of the different groups were selected for a phylogenetic study based on the ITS rDNA region (ITS1-5.8S-ITS2). Combining morphological, cultural, molecular and pathogenicity data, three species were distinguished. Phomopsis amygdali was shown to be the main pathogen on almond and is epitypified in the present work. Diaporthe neotheicola is reported for the first time on this host. A third species represented by a single isolate could not be unequivocally identified.