Abstract: Peronospora tabacina Adam, a downy mildew fungus, is a devastating disease of tobacco and a pathogen of import prohibition in Taiwan. For quarantine purpose, we developed a nested PCR method to detect this pathogen. With universal primer pair ITS1/ITS4, internal transcribed spacer (ITS) region of pathogen`s rDNA was amplified. Specific PCR primers (PT1/PT2) were designed based on ITS sequence and used to amplify a 493-bp rDNA fragments from P. tabacina. In order to increase the sensitivity, another primer pair (PT3/PT4) was used in a one-tube nested PCR. The expected 243-bp fragment was amplified from P. tabacina, but not from any other tested fungi or commercial tobacco leaves. The detection limit of this nested PCR could be low as 1 pg of template DNA. The present nested PCR method showed to be rapid, simple and available as a tool to screen P. tabacina infection for quarantine purpose.