Genetic incompatibility occurs when a mismatched pair of immune components triggers autoimmunity in hybrid plants. Highly diversified NLR receptors are key culprits of this genetic conflict, recognizing host proteins from different origins as immune triggers. Here, we report the molecular mechanism underlying DANGEROUS MIX (DM)-mediated autoimmunity in Arabidopsis thaliana, comprising DM2h/RPP1 NLR and its incompatible partner DM3, an alpha/beta hydrolase. Cryoelectron microscopy reveals the oligomeric nature of two natural DM3 variants in a trimer-of-dimers configuration. The polymorphism triggering autoimmunity is located at the dimer interface, resulting in drastic structural differences such that the dimerizing helix and loop reinforcing the interface are lost and disordered. Structure-function analysis shows that integrity of the dimer interface, but neither maintenance of hexamer nor its enzymatic activity, is the key factor for autoimmunity. Our finding pinpoints checkpoints embedded in the oligomeric configuration of a host enzyme that controls the switching mechanism of NLR activity.