Abstract: OBJECTIVE To identify DNA barcodes of bird's nest to provide molecular basis for the substrate of bird's nest. METHODS Mitochondria cytochrome oxidase subunit I gene(COI) fraction from 25 bird's nest samples was amplified by PCR method and sequenced. Then the intraspecific variation among the bird's nest samples and interspecific variation between bird's nest samples and fakes were analysed. Furthermore, the Barcoding Gap was evaluated by using Taxon DNA software. RESULTS the intraspecific variation among swiftlet was small. While many variation loci were found in different swiftlet species, and the genetic distance between species was significantly greater than the intraspecific genetic distance. Phylogenetic tree was constructed, in which the bird's nest samples from different sources were clustered together, while the fakes were distinguished from the bird's nest samples. CONCLUSION DNA barcode technology based on COI sequence can be used to distinguish the original source of bird's nest and its confounding products.