The aim of this work was the application of previously developed and development of new PCR markers for the evaluation of sunflower resistance to downy mildew. Twenty sunflower inbred lines were investigated. Plant resistance to downy mildew was determined by the whole seedling immersion method. Genomic DNA was extracted from the first pair of leaves, and its polymorphism was investigated by RAPD, SSR and several published markers for disease resistance. The presence of the markers Ha-NBS 7/R, Ha-NBS 8/R, Ha-NBS 9/R, in Pl6 donor lines (Ha-335, JM-8) and in resistant progeny (Ha-26, G12, G10, G11) confirm that HAP3 could be useful for the detection of the Pl6 gene. DNA polymorphism, which coincided with disease resistance, was revealed with one RAPD (UBC 119 fragment 900-1000 bp) and one SSR primer (ORS37 fragment 600-700 bp). Amplified fragments segregated in the same way, i.e., they appeared in 50% of the resistant genotypes. The non-expecting SSR fragment was purified, cloned and sequenced. The results indicated that this fragment is not a part of a coding sequence. Specific primers for the ampli fication of this fragment have been designed and the investigation of the inher itance of this SCAR marker is under way. None of the applied markers appeared in all resistant genotypes. In order to select lines for making crosses for use in further investigation, the obtained results were also used for the calculation of genetic distances between genotypes (simple matching coefficient) and the construction of a dendrogram (UPGMA method).