<p>Intermittent ponds of the New Jersey Pinelands are valuable ecosystems that support a diverse array of organisms including protists that play key roles in nutrient cycling and energy transfer within food webs. We used 18S-V9 DNA metabarcoding to explore protistan assemblages and their variation along a human-impact gradient in 30 natural and man-made Pinelands ponds. Heterotrophic protists were more diverse and abundant than phototrophs, especially in ponds surrounded by forest. Ciliates were the most abundant, while both ciliates and cercozoans were the most diverse taxonomic groups. Other prominent groups included amoebozoans, apicomplexans, peronosporomycetes, ochrophytes, dinoflagellates, and green algae. Protistan assemblage composition varied primarily with pH and along the land cover – eutrophication gradient. The most notable pattern in assemblage composition was a higher abundance of large-celled green algae in stormwater basins − man-made ponds with relatively low water quality, typically found in residential areas. While protistan assemblages in pond sediments were more diverse than those on submerged plants or artificial substrates, the latter still reflected the same environmental gradients as natural microhabitats.</p>