RNA sequencing (RNA-seq) was used for single nucleotide polymorphism (SNP) identification from two economically important obligate plant pathogens, Pseudoperonospora cubensis and P. humuli. The overall purpose of this study was to collect and utilize genomic data to further investigate the genetic differentiation of these two closely related species. The characterization of genes containing SNPs differentiating these two closely related downy mildew species may contribute to the development of improved detection and diagnosis strategies and improve our understanding of host specificity pathways.