Since the 1990s, a new Phytophthora species hybrid has been jeopardizing the natural population of alders throughout Europe. This new Phytophthora, P. alni, has been suggested as a natural hybrid between two closely related species of Phytophthora. Little is known about the epidemiology of this pathogen, because its direct isolation is not always satisfactory. In this study we developed three pairs of Polymerase Chain Reaction (PCR) primers derived from Sequence Characterized Amplified Regions (SCAR) that allow discrimination among the three subspecies of P. alni: P. alni subsp. alni, P. alni subsp. uniformis and P. alni subsp. multiformis. These molecular tools were successfully used to detect P. alni directly in different substrates such as infested river water and soil, and necrotic alder bark, without the need for any prior baiting or isolation stages. An Internal Amplification Control (IAC) was included to help discriminate against false negative samples due to the potential presence of inhibitory compounds in DNA extracts. These molecular tools should be useful for epidemiological studies on this emerging disease.