The obligate parasitic fungus-like organism Plasmopara halstedii (Farl.) Berl. et De Toni, is the causal agent of downy mildew disease in sunflower (Helianthus annuus). New races of this economically important parasite are regularly detected throughout the world. In addition, fungicide-resistant isolates have been reported in Europe and North America. These observations of parasite evolution, as well as the risk of propagation of the disease by infected seeds, means that it is necessary to guarantee the absence of Plasmopara halstedii in seed shipments. We report here the development of a rapid assay that can be used to detect infection by Plasmopara halstedii in plant tissues. Based on the nucleotide sequence information obtained from one cloned random amplified polymorphic DNA fragment, specific oligonucleotides were designed and used as primers for in vitro DNA amplification by polymerase chain reaction. An amplification product was detected on agarose gel stained with ethidium bromide when DNA from various Plasmopara halstedii races was tested, whereas no amplified DNA was detected when DNA from other origins was tested, including DNA from the host plant. The sensitivity of the technique was evaluated. The assay successfully reveals the presence of Plasmopara halstedii in infected sunflower plants prior to sporulation.