The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a serious quarantine pest first detected in Portugal and Europe in 1999. It is the causal agent of pine wilt disease (PWD). A resistance breeding programme has been initiated to contribute to control the evolution of the disease. Five hundred and four adult maritime pine, Pinus pinaster, trees were phenotypically selected as candidate trees for this programme from an area affected by PWD. To identify tolerance to the nematode, the selected trees were monitored monthly. Over the course of 1 year, 57 candidate trees died and were tested for the presence/absence of the PWN. As accuracy of detection is of major importance, an ITS-PCR-based method applied directly to wood from adult maritime pine trees was tested and compared with a standard morphological identification method. The results showed that the use of PCR to detect the pathogen provided more rapid and accurate results in comparison with the standard morphological identification. Thus, this method is suitable to be used in the survey of the breeding population for resistance/tolerance to PWD.