In an effort to assess the taxonomic identity of large-scale samplings of nematodes from the Konza Tallgrass Prairie, we sequenced a portion of the 18S rRNA gene and its associated internally transcribed spacer (ITS1) from 74 nematodes encompassing four taxonomic families. From these sequences, we have developed a series of molecular probes to distinguish 16 distinct microbivorous nematode taxa. Using a combination of low power microscopy and taxon-specific real-time probes, the 74 nematodes were correctly assigned to their respective taxonomic groups. This optimized method provides a high-throughput assay to determine nematode identities across larger data sets.