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Abstract Background Diet analysis is essential to understanding the functional role of large bird species in food webs. Morphological analysis of regurgitated bird pellet contents is time intensive and may underestimate biodiversity. DNA metabarcoding has the ability to circumvent these issues, but has yet to be done. Methods We present a pilot study using DNA metabarcoding of MT-RNR1 and MT-CO1 markers to determine the species of origin and prey of 45 pellets collected in Qinghai and Gansu Provinces, China. Results We detected four raptor species [Eurasian Eagle Owl (Bubo bubo), Saker Falcon (Falco cherrug), Steppe Eagle (Aquila nipalensis), and Upland Buzzard (Buteo hemilasius)] and 11 unique prey species across 10 families and 4 classes. Mammals were the greatest detected prey class with Plateau Pika (Ochotona curzoniae) being the most frequent. Observed Shannon’s and Simpson’s diversity for Upland Buzzard were 1.089 and 0.479, respectively, while expected values were 1.312 ± 0.266 and 0.485 ± 0.086. For Eurasian Eagle Owl, observed values were 1.202 and 0.565, while expected values were 1.502 ± 0.340 and 0.580 ± 0.114. Interspecific dietary niche partitioning between the two species was not detected. Conclusions Our results demonstrate successful use of DNA metabarcoding for understanding diet via a novel noninvasive sample type to identify common and uncommon species. More work is needed to understand how raptor diets vary locally, and the mechanisms that enable exploitation of similar dietary resources. This approach has wide ranging applicability to other birds of prey, and demonstrates the power of using DNA metabarcoding to study species noninvasively.