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Honey is a widely used natural product and the price of honey from Apis cerana (ACH) and A. dorsata (ADH) is several times more expensive than the one from A. mellifera (AMH), thus there are increasing fraud issues reported in the market by mislabeling or mixing honeys with different entomological origins. In this study, three species-specific primers, targeting the NADH dehydrogenase 2 (ND2) region of honeybee mitochondrial DNA, were designed and tested to distinguish the entomological origin of ACH, ADH, and AMH. Molecular analysis showed that each primer set can specifically detect the ND2 region from the targeted honeybee DNA, but not from the others. The amplicon size for A. cerana, A. dorsata and A. mellifera were 224, 302, and 377 bp, respectively. Importantly, each primer set also specifically produced amplicons with expected size from the DNA prepared from honey samples with different entomological origins. The PCR adulteration test allowed detection of 1% of AMH in the mixture with either ACH or ADH. Furthermore, real-time PCR and melting curve analysis indicated the possible discrimination of origin of honey samples. Therefore, we provide the newly developed PCR-based method that can be used to determine the entomological origin of the three kinds of honey.