We describe polymerase chain reaction (PCR) primers for gut analysis of aphid predators. The primers amplify aphid mitochondrial COII fragments ranging in size from 77 to 386 bp. Using these primers, we were able to distinguish six species of US Great Plains cereal aphids, including two congeners, Rhopalosiphum maidis (Fitch) and R. padi (L.), and to detect them in extracts of coccinellid and chrysopid predators. We devised a protocol for deriving half-lives of detectability for the DNA of a single aphid consumed by predators maintained under simulated field dietary and temperature conditions. Using this protocol and primers that amplify a 198-bp fragment, we determined statistically different half-lives of detectability for a single R. maidis of 3.95 h in Chrysoperla plorabunda (Fitch) and 8.78 h in Hippodamia convergens Guerin. The detectability half-life for a 339-bp R. maidis fragment was statistically longer in C. plorabunda but not in H. convergens. The sensitivity of the assay for the 198-bp fragment is 10−7 aphid equivalents. For species-specific predator gut analysis, PCR is superior to monoclonal antibody technology, giving comparable detectability half-lives with lower expense, much shorter development times, and greater certainty of a successful outcome.