NX toxins have been described as a novel group of type A trichothecenes produced by members of the Fusarium graminearum species complex (FGSC). Differences in structure between NX toxins and the common type B trichothecenes arise from functional variation in the trichothecene biosynthetic enzyme Tri1 in the FGSC. The identified highly conserved changes in the Tri1 gene can be used to develop specific PCR-based assays to identify the NX-producing strains. In this study, the sequences of the Tri1 gene from type B trichothecene- and NX-producing strains were analyzed to identify DNA polymorphisms between the two different kinds of trichothecene producers. Four sets of Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) methods were successfully developed to distinguish the common type B trichothecene producers and NX producers within FGSC. These promising diagnostic methods can be used for high-throughput genotype detection of Fusarium strains as a step forward for crop disease management and mycotoxin control in agriculture. Additionally, it was found that the Tri1 gene phylogeny differs from the species phylogeny, which is consistent with the previous studies.