A rapid and accurate diagnostic method is needed to detect asymptomatic carrier trees to assist in exterminating pine wilt disease caused by the pathogenic pine wood nematode (PWN), Bursaphelenchus xylophilus. We extracted DNA by the CTAB method from pine trees located in two natural stands, a Japanese black pine stand and a Japanese red pine stand, known to be infected with PWN, and detected the PWN by the nested-PCR method. Many trees of both pine species harboured PWN, although some of them had no external and/or internal symptoms such as yellowing of needles and disrupted resin exudation. Some Japanese black pine trees remained asymptomatic during the study and survived for 1 or more years after PWN infection without displaying any wilting symptoms. The diagnostic method adopted in the present study is useful and sensitive enough for practical use to detect the asymptomatic carrier trees and may prove to be an effective method to assist in control of the pest in the field.