PCR-based techniques to investigate predator-prey trophic interactions are starting to be used more widely, but factors affecting DNA decay in predator guts are still poorly understood. Here, we investigated the effects of time since feeding, temperature and amplicon size on the detectability of prey DNA in the gut content of two closely related predator species. Cereal aphids, Sitobion avenae, were fed to the carabid beetles Pterostichus melanarius and Nebria brevicollis. Beetles were allowed to digest their meal at 12°C, 16°C and 20°C, and batches of beetles were subsequently frozen at time periods from 0–72 h after feeding. Aphid DNA was detected within beetles' gut contents using primers amplifying fragments of 85, 231, 317 and 383 bp. Prey DNA detection rates were significantly higher in N. brevicollis than in P. melanarius, indicating fundamental dissimilarities in prey digestion capacities. High temperatures (20°C) and large amplicons (383 bp) significantly decreased detection rates. The shortest amplicon gave the highest prey DNA detection success, whereas no differences were observed between the 231 bp and the 317 bp fragment. Our results indicate that factors such as ambient temperature, predator taxon and amplicon size should all be considered when interpreting data derived from PCR-based prey detection. Correction for such factors should make calculation of predation rates in the field more accurate and could help us to estimate when predation events occur in the field.