To provide theoretical basis for the traceability and quality evaluation of edible bird's nests (EBNs), the Cytb sequence was applied to identify the origin of EBNs. A total of 39 experiment samples were collected from Malaysia, Indonesia, Vietnam and Thailand. Genomic DNA was extracted for the PCR reaction. The amplified products were sequenced. 36 sequences were downloaded from Gen Bank including edible nest swiftlet, black nest swiftlet, mascarene swiftlet, pacific swiftlet and germain's swiftlet. MEGA 7.0 was used to analyze the distinction of sequences by the method of calculating the distances in intraspecific and interspecific divergences and constructing NJ and UPMGA phylogenetic tree based on Kimera-2-parameter model. The results showed that 39 samples were from three kinds of EBNs. Interspecific divergences were significantly greater than the intraspecific one. Samples could be successfully distinguished by NJ and UPMGA phylogenetic tree. In conclusion, Cytb sequence could be used to distinguish the origin of EBNs and it is efficient for tracing the origin species of EBNs.