The tennite genus Reticulitermes species are serious pests of wooden structures and important quarantine insects in the world. It is very difficult to distinguish different Reticulitermes species based on their similar morphology, which brings much trouble to quarantine work for invasive Reticulitermes species. Here, we used the four molecular markers, 18 S rDNA, COI, COII and ITS genes, to carry out phylogenetic analysis and identification of the six Reticulitermes species. We found that R. labralis, R. speratus, R. aculabialis, R. chinensis and R.flaviceps from China clustered together in the phylogenetic tree of COI gene, consisting with geographical distribution of them. We suggest that COI gene is suitable for interspecific phylogenetic analysis of Reticulitermes species. Subsequently, we designed and synthesized specific primer and TaqMan probe based on the COI gene of R. chinensis, and found that this primer can specifically detect R. chinensis. The minimum concentration of DNA sample is lpg/μL for the identification of R. chinensis. Our results suggest that the rapid and accurate identification of different Reticulitermes species can be achieved by fluorescent PCR technology based on COI gene.