Important stem and grain diseases of soybean ( Glycine max [L.] Merr.) caused by Diaporthe aspalathi, D. caulivora, and D. longicolla reduce yield in the United States. Sources of resistance to these pathogens have previously been reported; however, there is limited information regarding their resistance when exposed to geographically distinct isolates of the same species. In this study, four accessions from the USDA Soybean Germplasm Collection with reported resistance to D. aspalathi, D. caulivora, or D. longicolla were evaluated using geographically representative isolates within each species from the United States. For each fungus, a greenhouse experiment was conducted as a completely randomized design with a factorial arrangement (isolate × accession). Plants were inoculated at the second to third trifoliate growth stage using toothpicks infested with isolates of each Diaporthe species. Pathogenicity was assessed 21 days postinoculation as 0 = no lesion, 0.5 = lesion length > 1 cm, and 1 = dead plant. A significant isolate-by-accession interaction ( P < 0.05) was observed to affect pathogenicity as analyzed using nonparametric statistics (relative treatment effects [RTEs]), indicating that accessions responded differently to the isolates. Correlation analyses suggested that the RTEs on ‘Tracy-M’, ‘Dowling’, and ‘Crockett’ were weakly to moderately correlated with those of the D. aspalathi-susceptible ‘Bragg’, as well as for ‘PI567473B’ and ‘Century’ ( D. caulivora), and ‘PI417507’ ( D. longicolla), with the RTEs on ‘Hawkeye’ ( P > 0.05) indicating possible genetic variation for resistance within these accessions. Our results provide information related to the resistance of previously identified accessions to develop commercial cultivars with resistance to important pathogens within the genus Diaporthe. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .