Stone fruit trees (Prunus spp.) are economically important fruit trees cultivated in South Africa. These trees are often grown in close proximity to vineyards and are to a large extent affected by the same trunk disease pathogens as grapevines. The aim of the present study was to determine whether stone fruit trees are inhabited by Diatrypaceae species known from grapevines and whether these trees could act as alternative hosts for these fungal species. Isolations were carried out from symptomatic wood of Prunus species (almond, apricot, cherry, nectarine, peach, and plum) in stone fruit growing areas in South Africa. Identification of isolates was based on phylogenetic analyses of the internal transcribed spacer region and β-tubulin gene. Forty-six Diatrypaceae isolates were obtained from a total of 380 wood samples, from which five species were identified. All five species have also been associated with dieback of grapevine. The highest number of isolates was found on apricot followed by plum. No Diatrypaceae species were isolated from peach and nectarine. Eutypa lata was the dominant species isolated (26 isolates), followed by Cryptovalsa ampelina (7), Eutypa cremea (5), Eutypella citricola (5), and Eutypella microtheca (3). First reports from Prunus spp. are E. cremea, E. citricola, and E. microtheca. Pathogenicity tests conducted on apricot and plum revealed that all these species are pathogenic to these hosts, causing red-brown necrotic lesions like those typical of Eutypa dieback on apricot.