Phomopsis stem canker of sunflower (Helianthus annuus L.) is a yield-limiting disease in the U.S. states of Minnesota, Nebraska, North Dakota, and South Dakota. Between the two causal fungi, Diaporthe gulyae and D. helianthi, D. gulyae is a new pathogen of sunflower. In this study, 205 accessions were selected from the cultivated sunflower germplasm and screened for resistance to a single isolate of D. gulyae using the mycelial-contact inoculation method in the greenhouse. The confection inbred ‘HA 288’ was used as the susceptible check. At V4 to V6 (four to six true leaves) growth stage, a 6-mm-diameter plug of potato dextrose agar infested with D. gulyae was placed in contact with the stem of the plants at the fourth node and covered with petroleum jelly. After inoculation, the sunflower plants were misted for 2 minutes every 2 h for 3 days. Fourteen days after inoculation, disease severity was evaluated on a 0 to 5 rating scale. Disease data were analyzed using non-parametric statistics and expressed as relative treatment effects (RTE). Of the 205 accessions, 41 accessions were determined to be significantly less susceptible to D. gulyae when compared to ‘HA 288’. The findings in this study suggest that the 41 accessions could be useful resources to breed for resistance to D. gulyae. Future studies will focus on the identification of single nucleotide polymorphism markers associated with D. gulyae resistance using genetic mapping strategies.