Sclerotinia crown and stem rot (SCSR) incited by Sclerotinia trifoliorum Eriks. causes severe losses in some fall-seeded, no-tillage plantings of alfalfa (Medicago sativa IL.) in Virginia. A mycelial plug inoculation technique was used to detect differences between cultivar (cv) responses of two alfalfa cvs, Arc and Vertus, under greenhouse conditions. A six dia plug from the margin of a 5-day-old culture of S. trifoliorum was placed near the crown area of a plant and incubated for a pre-determined period in a dew chamber at 18 C and 100% RH. Differences in isolate virulence were detected; cv Vertus was less susceptible than Arc to the less virulent isolates while the more virulent isolate (TAL 4) was equally severe on both. An incubation period of 96 hr produced significantly higher disease severity than 72, 48 or 24 hr, however, cv differentiation was best after 72 hr. Eight-, and nine-week-old plants were found to be most suitable for cv evaluation tests since younger seedlings were severely damaged and more mature plants did not develop sufficient symptom expression. Evaluation of twelve cvs with the virulent isolate (TAL 4) and the less virulent isolate (LAL 3) after 96 hr incubation produced significant differences between the mean disease severity ratings (MDSRs). Disease severity increased up to 20 days and then stabilized. Cultivar Anstar followed by WL 320, Vertus and Saranac AR were less susceptible in a majority of the tests; Endure and Euver performed well in some tests while Pioneer Brand 526 and Raidor performed poorly in all tests. This inoculation technique may act as the primary step in the selection of disease resistant germplasm for propagation, re-evaluation, and mass selection before field testing.