Over the past several years, southern corn rust (SCR) outbreaks caused by the fungus Puccinia polysora have become increasingly problematic for corn growers in the United States. SCR is currently diagnosed through the visual examination of disease symptoms and pathogen morphology, including pigmentation, size, shape, and location of fruiting structures. However, these characteristics are similar to those produced by the common corn rust fungus P. sorghi, confounding accurate visual diagnosis of SCR. Here we report the development of a realtime polymerase chain reaction assay that discriminates between P. polysora and P. sorghi. Sequences of the rDNA internal transcribed spacer region were determined for P. polysora and P. sorghi. 5-Carboxyfluorescein fluorophore-labeled hydrolysis probes that differed at 14 nucleotide positions between the species were developed from these data and used to screen DNA extracted directly from rust-infected corn leaves. Species-specific, reproducible identifications of the pathogens were made from as little as 50 pg of DNA within 30 min, and were reliably performed from both recent collections and herbarium specimens. This assay will be useful for rapid and accurate diagnosis of SCR, and could serve as a tool to monitor the distribution and incidence of the disease in the United States.