Phomopsis stem canker of sunflower is caused by two fungal pathogens, Diaporthe helianthi and Diaporthe gulyae, in the United States. In this study, two quantitative PCR (qPCR) assays were developed to detect and quantify D. helianthi and D. gulyae in sunflower. The two assays differentiated the two fungi from each other, other species of the genus Diaporthe, and pathogens, and they have high efficiency (>90%). The qPCR assays detected the two pathogens on plant samples exhibiting Phomopsis stem canker symptoms sampled from commercial sunflower fields in Minnesota, Nebraska, North Dakota, and South Dakota. Furthermore, the assays were used to screen cultivated sunflower accessions for resistance to D. helianthi and D. gulyae. The disease severity index (DSI) of the accessions significantly correlated (P < 0.0001) with the amount of pathogen DNA from the qPCR assays. The qPCR assays identified PI664232 and PI561918 to be significantly less susceptible (P ≤ 0.05) to D. helianthi and D. gulyae, respectively, when compared with the susceptible check cultivar HA 288, and this was in agreement with the DSI. These results suggest that the qPCR assays for D. helianthi and D. gulyae can be used as a reliable tool to diagnose Phomopsis stem canker and screen sunflower germplasm for disease resistance.