Melampsora medusae (Mm), one of the causal agents of poplar rust, is classified as an A2 quarantine pest for European Plant Protection Organization (EPPO) and its presence in Europe is strictly controlled. Two formae speciales have been described within Mm, Melampsora medusae f. sp. deltoidae (Mmd), and Melampsora medusae f. sp. tremuloidae (Mmt) on the basis of their pathogenicity on Populus species from the section Aigeiros (e.g. Populus deltoides) or Populus (e.g. Populus tremuloides), respectively. In this study, a real-time polymerase chain reaction (PCR) assay was developed allowing the detection of Mmd, the forma specialis that is economically harmful. A set of primers and hydrolysis probe were designed based on sequence polymorphisms in the large ribosomal RNA subunit (28S). The real-time PCR assay was optimized and performance criteria of the detection method, i.e. sensitivity, specificity, repeatability, reproducibility, and robustness, were assessed. The real-time PCR method was highly specific and sensitive and allowed the detection of one single urediniospore of Mmd in a mixture of 2 mg of urediniospores of other Melampsora species. This test offers improved specificity over currently existing conventional PCR tests and can be used for specific surveys in European nurseries and phytosanitary controls, in order to avoid introduction and spread of this pathogen in Europe.