Ukraine is one of the world’s leaders in production of the sunflower seeds. Increasing the cultivated areas with sunflower in Ukraine led to spreading of the multiple pathogens infection, including Downy mildew. Developing hybrids, resistant to causal pathogen of Downy mildew (Plasmopara halstedii (Farl.) Berl. & de Toni), is one of the critical tasks in sunflower breeding. Breeding process includes field- and lab-tests of the initial material for resistance. Molecular markers have advanced breeding practice in the past decades, however there are still unmet needs for reliable high-throughput selection of the pathogen resistant starting material and differentiation of the plants infected by different pathogens. The aim of this study was to demonstrate the possibility of usage of specific sequence of Plasmopara halstedii genome as DNA marker for detection of pathogen in seedlings and tissues of sunflower plant. CTAB protocol was used for DNA isolation from the sunflower seedlings, the leaves of flowering plants with chlorosis and Plasmopara halstedii sporangium removed from infected samples. PCR was performed with primers PHAL-F and PHAL-R. We tested the known DNA marker (310 bp fragment from ribosomal DNA of Plasmopara halstedii) for detection of pathogen in different tissues of sunflower plant and at different stages of plant development. To accomplish molecular testing, we collected 60 seedlings when they were lab-tested and visible sign of Plasmopara halstedii infection started to occur. PCR-test results coincided with standart lab-test results. Specified DNA marker was re-validated in the total DNA, isolated from sporangium as well as from seedlings of infected pathogen resistant/susceptible inbred lines of Ukrainian breeding and 60 F2 crosses. An independent set of field grown plants with unknown resistance to Plasmopara halstedii, having symptoms of the bacterial/fungal/viral pathology were used for marker screening. The genotypes, infected with downy mildew, were successfully identified. Pathogen appeared to be concentrated in the vessels of sunflower leaves, in contrast to parenchymal tissue. DNA-test might be useful to control the degree of contamination of sunflower plants with Downy mildew spores in the field, especially for diagnosis of the hidden disease. Our study demonstrates that PCR-test could be an addition to the lab-test of seedlings or an independent test of mature plants, grown in the field.