Diaporthe canker of pome and stone fruit trees, caused by Diaporthe ambigua, is a newly recognized disease in the deciduous fruit growing areas of South Africa. A Potential biological control strategy for Diaporthe canker would make use of viruses or virus- like agents capable of debilitating D. ambigua. Hypovirulence-associated double-stranded RNA (dsRNA) was isolated from a number of different D. ambigua strains. In all cases a single dsRNA segment was observed. This is in contrast to the related Cryphonectria  parasitica where it is more common to find many different sizes of dsRNA. The D. ambigua dsRNA segments showed similar electrophoretic mobility in agarose gels, and the size was estimated to be approximately 4.3 kb. Hybridization between the different dsRNAs showed a high degree of homology. However, no hybridization was evident between the dsRNA in D. ambigua and C. parasitica even though the hybridization stringency was decreased in an attempt to reveal possible homology. The presence of D. ambigua strains in a localized area, carrying a single dsRNA segment of similar size and homology suggests that natural spread of dsRNA within the D. ambigua population has occurred. The lack of homology between dsRNA from South African hypovirulent strains of D. ambigua and dsRNA from North American and European hypovirulent strains of C. parasitica suggests different origins.