A polymerase chain reaction (PCR)-based assay was developed for the specific detection of the fungal pathogens Diplodia pinea and D. scrobiculata from pine host tissues. Variation among mitochondrial small subunit ribosome gene (mt SSU rDNA) sequences of Botryosphaeria species and related anamorphic fungi was exploited to design primer pairs. Forward primer DpF and forward primer DsF, each when used with the nonspecific reverse primer BotR, amplified DNA of D. pinea or D. scrobiculata, respectively. Specificity was confirmed using multiple isolates of each of these two species and those of closely related fungi including Botryosphaeria obtusa. The detection limits for DNA of each pathogen in red and jack pine bark were 50 to 100 pg μl-1 and 1 pg μl-1 in red and jack pine wood. The assay was tested using naturally occurring red and jack pine seedlings and saplings exhibiting symptoms of Diplodia collar rot. Samples from lower stems/root collars of 10 dead trees of each species from each of three sites at each of two locations were tested. Results were positive for D. pinea or D. scrobiculata for the large majorities of symptomatic bark and wood samples from both locations. For positive samples, however, there were effects of location and host species on detection of D. pinea (more frequent on red pine) and D. scrobiculata (more frequent on jack pine) (P < 0.01 in both cases). These results indicate that these new primers are potentially useful for studies in areas or hosts in which both pathogens may be present.