The purpose of our study was to survey the freshwater planktonic protists within an inland natural preserve in the Ridge and Valley physiographic province of the Appalachian Region using metabarcoding. Microbial eukaryotes are essential primary producers and predators in small freshwater ecosystems, yet they are often overlooked due to the difficulty of identification. This has been remedied, in part, by the cost reduction of high throughput DNA sequencing and the growth of barcode databases, making the identification and analysis of microorganisms by way of metabarcoding surveys in complex ecosystems increasingly feasible. Water samples were collected from five sites at the Natural Tunnel State Park in Scott County, VA (USA), representing three common bodies of water found in this region. Samples were initially collected during a Bioblitz event in April 2016 and then seven and fourteen weeks afterwards. Metabarcode analysis of the 23S and 18S genes identified 3663 OTUs representing 213 family level and 332 genus level taxa. This study provides an initial barcode census within a region that has a reputation as a temperate biodiversity “hotspot”. The overall protist diversity was comparably high to other temperate systems, but not unusually high; the microalgal diversity, however, was higher than that reported for other temperate regions. The three types of water bodies had their own distinctive protist biomes despite close proximity.