Morphological identification of prey fragments in vampire bat feces is impossible because of an exclusively blood-based diet. Therefore, studies of their foraging ecology require innovative approaches. We investigated the diet of Desmodus rotundus using a PCR-restriction fragment length polymorphism (RFLP) molecular method by amplifying the cytochrome b mitochondrial gene (380 bp) from DNA fecal samples collected from captive bats fed with blood from chickens, cattle, pigs, dogs, and humans—the 5 most frequently attacked prey species in rural areas of the Brazilian Amazonia. The prey preference of the vampire bat was investigated in 18 riverine villages, where the availability of domestic animals to bats was quantified. Prey DNA amplified from fecal samples exhibited no visible signals of vampire bat DNA. A PCR—RFLP flowchart and a combination of 2 DNA restriction enzymes allowed the direct identification of prey to species level. The enzymes' restriction profile did not overlap with those of vampire bats or wild mammal and avian species. Chickens were the most attacked prey species (61.4% of the identifications, n = 27), but pigs were highly preferred in relation to prey availability. This suggests a preference for mammalian blood in D. rotundus diet, with chickens exploited as a secondary food source. No wild vertebrate species was identified in the fecal samples, indicating that vampire bats are selectively feeding on the blood of domesticated animals, probably because they are more predictable and easily accessed resources.