Mastication, digestion, and rapid evacuation rates make visual identification of larval fish remains in the digestive tracts of predatory fishes problematic. Recent advances in molecular technology, however, have increased the likelihood of identifying remnants of partially digested larval prey, thereby enabling assessment of predator impacts on local populations. Conducting controlled laboratory experiments, we evaluated the utility of quantitative polymerase chain reaction (qPCR) for identification of Razorback Sucker Xyrauchen texanus larvae in the digestive tracts of Green Sunfish Lepomis cyanellus and Western Mosquitofish Gambusia affinis. Primers and a probe were developed to amplify a fragment from Razorback Sucker mtDNA. Tests using a suite of potential predators and prey indicated that these primers and probes amplified only mtDNA from Razorback Sucker and Flannelmouth Sucker Catostomus latipinnis, an allopatric and allochronic species in the lower Colorado River. Amplification with primers for Razorback Sucker-specific DNA fragments identified Razorback Sucker DNA in the digestive tracts of 87.5% of Green Sunfish and Western Mosquitofish at 2 h postfeeding. After 12 h, DNA fragments were identified in 75.0% of Green Sunfish but in only 28.6% of Western Mosquitofish. No Razorback Sucker DNA was found in 24-h postfeeding samples, though it was detected in 12.5% of both Western Mosquitofish and Green Sunfish at 48 h postingestion. The sensitivity of qPCR provides a useful tool for extending the time period in which Razorback Sucker larvae can be identified beyond that of traditional visual analyses of stomach contents.