Tilletia controversa is an internationally quarantined pathogenic fungus that causes dwarf bunt of wheat and is similar to Tilletia caries in both teliospore morphology and genetic structure. This study developed a rapid and sensitive immunofluorescence method for differentiating the teliospores of T. controversa from T. caries.The method utilizes monoclonal antibody D-1 against teliospores of T. controversa as well as a PE-Cy3-conjugated goat anti-mouse antibody (overlapping light excitation of 495 and 555 nm). The orange cycle fluorescent signal was stronger against T. controversa teliospores in the outer spore wall and net ridge, whereas only the green signal was observed for the protoplasm of T. caries teliospores. The detection limit of this method was 2.0 μg ml(-1) of the D-1 monoclonal antibody.This study describes the production and diagnostic application of a novel mouse monoclonal antibody specific to T. controversa teliospores.This method could be used for the on-site identification of T. controversa teliospores in the near future and will help in selecting fungicides to control dwarf bunt of wheat as further technical developments are achieved.