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<div>Phomopsis stem canker of sunflower (<i>Helianthus annuus</i>) is a priority disease of the sunflower farmers in the United States. The disease is predominantly caused by <i>Diaporthe helianthi</i> and <i>Diaporthe gulyae</i>. The objective of this study was to use quantitative polymerase chain reaction (qPCR) assays to screen sunflower genotypes for resistance to <i>D. helianthi</i> and <i>D. gulyae</i>. Fifty-four genotypes that exhibited potential resistance to Phomopsis stem canker were selected from 288 Plant Introductions screened under natural disease pressure in a field trial. Sunflower cultivar ‘HA 288’ (PI 552934) was used as the susceptible check for the study. Each of the sunflower genotypes were inoculated with a <i>Diaporthe</i>-infested mycelial plug of an aggressive isolate of <i>D. helianthi </i>or <i>D. gulyae</i> in separate experiments under greenhouse conditions. At 14 days after inoculation, the genotypes were rated for disease. Additionally, the amount of pathogen DNA in the inoculated plants was determined using qPCR assays specific to the two pathogens. For both the pathogens, the susceptible genotypes had significantly higher disease severity and amount of pathogen DNA as compared to the resistant accessions. The resistant genotypes identified in this study will be useful for developing commercial sunflower hybrids with resistance to<i> D. helianthi</i> and <i>D. gulyae</i>.</div>