Phylogenetic analysis of the ITS rDNA region was carried out with two economically important downy mildews, Pseudoperonospora cubensis, which infects species of Cucumis, Cucurbita, and Citrullus belonging to Cucurbitaceae, and P. humuli, which infects plants of the genus Humulus belonging to Cannabaceae. Two closely related species, P. cannabina and P. celtidis, were also included to reveal taxonomic relationships with the first two mildews. All four species formed a well-resolved clade when compared with the ITS sequences of other downy mildew genera, using Bayesian inference and maximum parsimony. The P. cubensis isolates obtained from different hosts and (or) geographical origins in Korea, exhibited no intraspecific variability in the ITS sequences. The phylogenetic analyses of P. cubensis and P. humuli showed that they share a high level of sequence homology; the morphology of the sporangiophores, sporangia, and dehiscence apparatus confirmed the similarity of the two species. We therefore reduce P. humuli to the status of a taxonomic synonym of P. cubensis.