Diplodia pinea and Diplodia scrobiculata are opportunistic pathogens associated with various disease symptoms on conifers that most importantly include die-back and stem cankers. Two viruses with dsRNA genomes, Sphaeropsis sapinea RNA virus 1 and 2 (SsRV1 and SsRV2) are found in D. pinea and an undescribed dsRNA element is known to occur in D. scrobiculata. We have partially characterized the putative RNA-dependent RNA polymerase (RdRp) of the undescribed dsRNA element and designed virus-specific primers from the RdRp regions of all three virus genomes. This made it possible to screen for the presence of the three viruses in a collection of D. pinea and D. scrobiculata isolates using real-time PCR. Triple infections with all three viruses occurred in D. pinea and D. scrobiculata. Co-infections with SsRV1 and SsRV2 were common but found only in D. pinea. Co-infection with SsRV1 and the undescribed dsRNA element was rare and observed only in D. pinea. Single infections with either SsRV1 or SsRV2 were equally common, while the undescribed dsRNA element never occurred alone. SsRV1 occurred alone in both D. pinea and D. scrobiculata while SsRV2 occurred alone only in D. pinea. There were only two instances where the undescribed dsRNA element was observed in D. pinea and it was otherwise found only in D. scrobiculata. This study highlights the complex interactions between the viruses found in the closely related plant pathogenic fungi, D. pinea and D. scrobiculata. It illustrates the importance of not only characterizing viruses infecting fungi but also of determining the interactions between mycoviruses and their fungal hosts.