Sunflower line with resistance for downy mildew (Ha-26R) and susceptible line (Ha-26S) were inoculated with the suspension of Plasmopara halstedii zoosporangia race 730 in the phase of first pair of leaves. The expression of defense related genes was investigated in the time period of 2 to 96 h after treatment. Several categories of defense-related genes: signalization (EDS1 and EDR1); H 2 O 2 producing (Hacaox and Haoxox); antioxidative response (SODc and SODp) pathogenesis related (chi PAL PR5) were examined. Most examined defense related genes were constitutive with higher expression in resistant line. However in response to secondary downy mildew infection six genes were upregulated. Upregulation of HaEDS1 signaling gene 2 h after infection indicates that SA mediated response is activated. Pathogenesis related genes: chitinase and PR5 were also upregulated in the earliest time point. Other defense related genes: SODp Caox and OxOx were sequentially upregulated from 4 to 48 h after infection in resistant line. Our results indicate that the early response of defense related genes to secondary downy mildew infection resembles to hypersensitive-like reaction and is connected with resistance conferred by Pl6 gene. According to our results resistance to secondary infection is characterized with earlier upregulation of PR5 in comparison to primary infection.