Sunflower leaf spot caused by the necrotrophic pathogen Alternaria helianthi is one of the most important biotic constraints in sunflower production. The biochemical basis of resistance to infection by A. helianthi in sunflower is poorly elucidated. Differential activation of early defense responses like increased peroxidase activity predisposes the host plant resistance. In the present study, the active role of peroxidase in resistant and susceptible interactions of sunflower with the leaf spot pathogen was demonstrated using native PAGE analysis and Northern blotting. Differential time-dependent induction of peroxidase isoforms was evident in resistant and susceptible genotypes after pathogen inoculation. In the resistant interaction, three peroxidase (PO) isoforms (PO1, PO2, and PO3) were found to be expressed at 2 and 12 h after inoculation, but in susceptible interaction, PO2 was present at 24 and 48 h and PO3 was found only at 24 h after inoculation. However, in the susceptible genotype, expression intensity was lower than in the resistant interaction at all the time intervals. Molecular expression profiling by Northern blotting using heterologous cDNA probes showed rapid up-regulation of peroxidase at 2 and 12 h in resistant interaction. These results demonstrate that peroxidase isoforms are involved in defense against A. helianthi, and genetic regulation in the form of transcript accumulation is important for expression of disease resistance.