Fusarium root rot of lisianthus (Eustoma grandiflorum) caused by Fusarium solani is one of the most important and damaging lisianthus diseases. It occurs commonly in Japan and worldwide and causes serious crop losses. However, little effort has been made to breed lisianthus for resistance to this disease. We initiated a breeding program for resistance to F. solani in 2014. Twenty-nine lisianthus cultivars (E. grandiflorum) and one inbred line of Eustoma exaltatum were evaluated for resistance to two isolates (MAFF712388 and MAFF712411) of F. solani, as a first step toward the breeding of resistant cultivars. Seedlings were inoculated following injury by needle, then grown using hydroponic equipment—an efficient and reliable method for evaluating resistance. We found large differences in resistance among the 29 cultivars and the one inbred line tested. ‘Papillon Pink Flash’ was highly resistant to both isolates and showed no disease symptoms in a total of four tests. Furthermore, E. exaltatum Ohkawa No. 1 was highly resistant to isolate MAFF712388, showing no disease symptoms, and resistant to isolate MAFF712411. On the other hand, ‘Mink’, ‘Nagisa A’, ‘Nagisa B’, and ‘Vulcan Marine’ were stably susceptible with 70% to 100% of plants of these four cultivars wilting in all tests. MAFF712411 had greater pathogenicity than MAFF712388, but it is not clear whether the two isolates belong to different races.