Fluopyram, a succinate dehydrogenase inhibitor (SDHI) fungicide, was recently registered for use as a soybean seed treatment for management of sudden death syndrome (SDS) caused by Fusarium virguliforme. Although registered and now used commercially, in vitro baseline fungicide sensitivity of F. virguliforme to fluopyram has not yet been established. In this study, the baseline sensitivity of F. virguliforme to fluopyram was determined using in vitro growth of mycelium and germination of conidia assays with two collections of F. virguliforme isolates. A total of 130 and 75 F. virguliforme isolates were tested using the mycelial growth and conidia germination assays, respectively, including a core set of isolates that were tested with both assays. In the mycelial growth inhibition assay, 113 out of 130 isolates (86.9%) were inhibited 50% by effective concentrations (EC50) less than 5 µg/ml with a mean EC50 of 3.35 µg/ml. For the conidia germination assay, 73 out of 75 isolates (97%) were determined to have an estimated EC50 of less than 5 µg/ml with a mean EC50 value of 2.28 µg/ml. In a subset of 20 common isolates that were phenotyped with both assays, conidia germination of F. virguliforme was determined to be more sensitive to fluopyram (mean EC50 = 2.28 µg/ml) than mycelial growth (mean EC50 = 3.35 µg/ml). Hormetic effects were observed in the mycelial growth inhibition assay as 22% of the isolates demonstrated more growth on medium amended with the lowest fluopyram concentration (1 µg/ml), as compared with the nonfluopyram amended control. It was not possible to determine EC50 values for nine out of 185 isolates (4.8%), as those isolates were not inhibited by 50% even at the highest fluopyram concentrations of 100 µg/ml for mycelial growth and 20 µg/ml for conidia germination inhibition assays. On the whole, the F. virguliforme population appears to be sensitive to fluopyram, and this study enables future monitoring of fungicide sensitivity.