Soybean is threatened by many pathogens that negatively affect this crop’s yield and quality, such as various Fusarium species that cause wilting and root rot diseases. Fusarium root rot (FRR) in soybean can be caused by F. graminearum and other Fusarium spp. that are associated with Fusarium head blight (FHB) in cereals. Therefore, it was important to inquire whether Fusarium pathogens from soybean can cause disease in wheat and vice versa. Here, we investigated the FRR complex in Manitoba (Canada) from symptomatic plants, using both culture- and molecular-based methods. We developed a molecular diagnostic toolkit to detect and differentiate between several Fusarium spp. involved in FHB and FRR, then we evaluated cross-pathogenicity of selected Fusarium isolates collected from soybean and wheat, and the results indicate that isolates recovered from one host can infect the other host. Trichothecene production by selected Fusarium spp. was also analyzed chemically via liquid chromatography mass spectrometry in both soybean (root) and wheat (spike) tissues. Trichothecenes were also analyzed in soybean seeds from plants with FRR to check the potentiality of trichothecene translocation from infected roots to the seeds. All of the tested Fusarium isolates were capable of producing trichothecenes in wheat spikes and soybean roots, but no trichothecenes were detected in soybean seeds. This study provided evidence, for the first time, that trichothecenes were produced by several Fusarium spp. (F. cerealis, F. culmorum, and F. sporotrichioides) during FRR development in soybean.