Next-generation sequencing technologies are revealing a more complete picture of biological diversity within various environments and are posed to fundamentally challenge our current understanding of species interactions. However, in order to maximize the potential of this new resource, appropriate molecular loci need to be chosen and vetted to facilitate comparison across studies. Here we evaluate the use of the mitochondrial COI barcode locus for estimating oomycete diversity in both soil and aquatic habitats via pyrosequencing. COI has several advantages over other markers, namely the ITS and LSU ribosomal RNA regions, used in studies of fungal and microbial eukaryote diversity; because COI encodes a translated product, sequencing errors such as indels can easily be detected. In addition, the conservation of COI allows for more robust sequence alignment and phylogenetic analysis across greater evolutionary distances, while still retaining enough nucleotide variation for species-level identifications. Our pyrosequencing results recovered more than twice the number of oomycete species compared to our culture-based approach (58 versus 25 species, respectively); in addition, we were able to identify 40 well supported, phylogenetically novel lineages, some of which may represent new species. As with all sequenced-based approaches, a number of important biases must be considered in order to realize the full potential these new and exciting technologies bring to the study of phytobiomes.