Puccinia horiana, the causal agent of chrysanthemum white rust, is a pathogen of quarantine status in many countries where Chrysanthemum × morifolium cultivars are grown. Historically, identification protocols for white rust relied upon macroscopic symptom development and microscopic examination of infected leaves for teliospores. Symptoms become visible 7 to 10 days after initial infection under favorable conditions followed by the production of telia. Infected plants can therefore evade detection before symptoms and fruiting bodies are evident. Conventional and real-time polymerase chain reaction (PCR) assays were developed to detect P. horiana using primers designed to amplify portions of the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA (rDNA). The species-specific primers could detect the pathogen from 1 ng of DNA isolated from infected leaf tissue in conventional PCR assays and from 1 pg in real-time PCR assays. While both assays were capable of detecting P. horiana in symptomatic tissue, the greater sensitivity offered by the real-time PCR assay makes it more reliable for detecting the pathogen during the latent stage of infection. The P. horiana primers did not amplify the rDNA target using DNA isolated from leaf tissue infected with P. chrysanthemi.