Developing hybrids resistant to causal pathogen of Downy mildew ( Plasmopara halstedii (Farl.) Berl. & de Toni) is one of the critical tasks in sunflower breeding. Molecular markers have advanced breeding practice in the past decades however there are still unmet needs for reliable high-throughput (HT) selection of the pathogen resistant starting material and differentiation of the plants infected by different pathogens. In this study we tested the known DNA marker (308 bp fragment from ribosomal DNA of P. halstedii ) for detection of pathogen in different tissues of sunflower plant and at different stages of plant development. 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 F 2 crosses. An independent set of field grown plants with unknown resistance to P. halstedii having symptoms of the bacterial/fungal/viral pathology were used for HT screening and genotypes infected with downy mildew were successfully identified. Pathogen appeared to be concentrated in the vessels of sunflower leaves in contrast to parenchymal tissue. Our study demonstrates an addition to whole seedling inoculation technique of P. halstedii detection which allows HT identification of the pathogen infected and non-infected sunflower plants.