Hop downy mildew (Pseudoperonospora humuli) and hop powdery mildew (Podosphaera macularis) are the most important pathogens of hop (Humulus lupulus). The early detection and identification of these pathogens are often made difficult by symptomless or combined infection with another pathogens. Molecular analysis of internal transcribed spacer (ITS) regions of rDNA is a novel and very effective method of species determination. Therefore, specific PCR assays were developed to detect the pathogens Pseudoperonospora humuli and Podosphaera macularis in naturally infected hop plants. The specific PCR primer combinations P1 + P2 and S1 + S2 amplified specific fragments from Pseudoperonospora humuli and Podosphaera macularis, respectively, and did not cross-react with hop DNA nor with DNA from other fungi. PCR primer combinations R1 + R2 and R3 + R4 could be used in multiplex PCR detection of Pseudoperonospora humuli, Podosphaera macularis, Verticillium albo-atrum and Fusarium sambucinum. Phylogenetic relationships were inferred for 42 species of the Erysiphales from nuclear rDNA (ITS1, 5.8S, ITS2). The molecular characterisation and phylogenetic analyses confirmed the species identification of hop powdery mildew. The PCR assays used in this study proved to be accurate and sensitive for detection, identification, classification and disease-monitoring of the major hop pathogens.