Oomycetes are a group of eukaryotes related to brown algae and diatoms, many of which cause disease in plants and animals. Improved methods are needed for rapid and accurate characterization of oomycete communities. Due to the unique order of tRNA coding regions flanking the mitochondrial rps10 gene, it was possible to design oomycete-specific primers that may be useful for oomycete metabarcoding. We evaluated the utility of this locus for use with metabarcoding by comparing reference sequences and sequencing a mock community and environmental samples using the Illumina MiSeq. The primers described herein are predicted to amplify all oomycetes based on available reference sequences and MiSeq sequences contained reads for 23 of the 24 species included in the mock community. Simulated PCR using reference databases and sequencing of environmental samples indicates the proposed rps10-based technique results in far less amplification of non-target organisms than the ITS1-based method. The portion of the rps10 gene amplified also has much more taxonomic resolution than ITS1, allowing a greater proportion of species to be distinguished.</p><p>We provide a new website with a rps10 reference database and all protocols relevant for oomycete metabarcoding.