The most economically important plant pathogens in the genus Pseudoperonospora (family Peronosporaceae) are Pseudoperonospora cubensis and P. humuli, causal agents of downy mildew on cucurbits and hop, respectively. Recently, P. humuli was reduced to a taxonomic synonym of P. cubensis based on internal transcribed spacer (ITS) sequence data and morphological characteristics. Nomenclature has many practical implications for pathogen identification and regulatory considerations; therefore, further clarification of the genetic and pathogenic relatedness of these organisms is needed. Phylogenetic analyses were conducted considering two nuclear and three mitochondrial loci for 21 isolates of P. cubensis and 14 isolates of P. humuli, and all published ITS sequences of the pathogens in GenBank. There was a consistent separation of the majority of the P. humuli isolates and the P. cubensis isolates in nuclear, mitochondrial, and ITS phylogenetic analyses, with the exception of isolates of P. humuli from Humulus japonicus from Korea. The P. cubensis isolates appeared to contain the P. humuli cluster, which may indicate that P. humuli descended from P. cubensis. Host-specificity experiments were conducted with two reportedly universally susceptible hosts of P. cubensis and two hop cultivars highly susceptible to P. humuli. P. cubensis consistently infected the hop cultivars at very low rates, and sporangiophores invariably emerged from necrotic or chlorotic hypersensitive-like lesions. Only a single sporangiophore of P. humuli was observed on a cucurbit plant during the course of the studies. Together, molecular data and host specificity indicate that there are biologically relevant characteristics that differentiate P. cubensis and P. humuli that may be obfuscated if P. humuli were reduced to a taxonomic synonym of P. cubensis. Thus, we recommend retaining the two species names P. cubensis and P. humuli until the species boundaries can be resolved unambiguously.