ABSTRACT Sphaeropsis sapinea is the causal agent of Sphaeropsis tip blight disease of pines. Past surveys of diseased and symptomless Austrian and Scots pines revealed that latent infections of symptomless shoots by S. sapinea are common. The role of these latent infections in the tip blight disease is unknown. A sampling technique and nested-polymerase chain reaction (PCR) protocol were developed to detect latent S. sapinea in symptomless pine shoots. The sampling protocol was designed to be minimally destructive to the shoot so it could be preserved for further studies. The primers that were developed were specific for S. sapinea DNA and did not amplify DNA from any of 13 other endophytic fungal species that were commonly isolated from symptomless pine shoots. The PCR primers also amplified DNA of Botryosphaeria obtusa, which was, however, rare in symptomless Austrian pine tissues. The protocol detected as little as 0.93 pg of S. sapinea DNA in terminal bud samples and 10.4 pg of DNA in bark samples. Correlation (chi-square) analyses indicated that the nested-PCR protocol detected latent S. sapinea infections in both bud and bark samples with an efficiency that was statistically equivalent to isolating the fungus from the tissue. The nested-PCR protocol will make it possible to more quickly identify latent S. sapinea infections in symptomless pine shoots and should be useful in future studies of the latency phenomenon.