Aims: Wheat (Triticum aestivum) is one of the most important crop species, but yields are often drastically reduced by rust epidemics. In this report, we describe a rapid and sensitive immunofluorescence method for the detection of urediniospores of the fungi Puccinia striiformis f.sp.tritici, Pucciniatriticina and Puccinia graminis f.sp.tritici, which are causal agents of wheat rust. Methods and Results: The method uses monoclonal antibody LPT-2 against the urediniospores of P.triticina and PE-cy3 goat anti-mouse. Urediniospores of P.triticina or those of two species that are difficult to distinguish from P.triticina, P.striiformis f.sp.tritici or P.graminis f.sp.tritici were immobilized on a glass slide, and the sample was then treated with LPT-2. Thereafter, a second antibody, goat anti-mouse conjugated PE-cy3, was added, and the slide was observed in a fluoroscope. The fluorescent signal was strong with P.triticina urediniospores, weak with P.striiformis f.sp.tritici urediniospores and weak-to-intermediate with P.graminis f.sp.tritici urediniospores. The detection limit of this method was 2ngml(-1) of the monoclonal antibody LPT-2. Conclusions: In this article, we describe the production and diagnostic application of a novel mouse monoclonal antibody specific to urediniospores of P.triticina. Significance and Impact of the Study: After further technical development, this method may become a tool for on-site identification of P.triticina urediniospores and will therefore help in the selection and timing of fungicide applications for control of wheat rust outbreaks.