Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is a serious disease of many cruciferous crops and frequently poses a threat to the sustainable and profitable production of these crops worldwide. Differences in seedling resistance to S. sclerotiorum across 46 diverse cruciferous genotypes from 12 different species were assessed by comparing the extent of pathogenesis on inoculated cotyledons under controlled conditions. Selections of Brassica carinata, B. incana, B. juncea, B. napus, and B. napus introgressed with B. carinata, B. nigra, B. oleracea, B. rapa var. rosularis, B. rapa var. chinensis, B. tournefortii, Raphanus raphanistrum, R. sativus, and Sinapis arvensis were tested. The average size of lesions on cotyledons 48 h post inoculation varied from 0.8 to 7.3 mm. The three most resistant genotypes with the smallest lesions were all from B. oleracea (viz., B. oleracea var. italica ‘Prophet’ and B. oleracea var. capitata ‘Burton’ and ‘Beverly Hills’). Representatives of R. raphanistrum, S. arvensis, B. juncea, and B. carinata were the most susceptible to S. sclerotiorum, with the largest lesions. To our knowledge, this is the first report of high levels of resistance to S. sclerotiorum in B. oleracea at the cotyledon stage and also the first report of the host cotyledon reactions against S. sclerotiorum for all tested species except B. napus and B. juncea. The mean lesion size for B. napus introgressed with B. carinata was 5.6 mm, which is midway between the lesion size for the two parent species B. napus (5.1 mm) and B. carinata (5.8 mm). Separate genetic control for cotyledon versus mature plant resistance was demonstrated by the lack of correlation between lesion size from S. sclerotiorum on the cotyledon with the severity of disease initiated by stem inoculation or natural processes in a previous field test. On the most resistant genotypes, B. oleracea var. italica Prophet and var. capitata Burton, growth of S. sclerotiorum on the cotyledon surface prior to penetration was severely impeded, production of appressoria inhibited, and both cytoplasm shrinkage and protoplast extrusion in S. sclerotiorum hyphae prevalent. This is the first report of such resistant mechanisms in B. oleracea. Genotypes with cotyledon resistance identified in this study will be of great value not only in furthering our understanding of resistance mechanisms across different cruciferous species but also could be exploited for developing commercial crucifer cultivars with high-level resistance against S. sclerotiorum.