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Background: Coptotermes formosanus Shiraki was accidentally introduced into the United States from Asia. The introduction of the pest has brought significant economic consequences. During the past decade, Mississippi has become a significantly infested state, partly due to the proximity to coastal port cities such as New Orleans. This study was initiated to investigate the origin and infestation route of C. formosanus in southern Mississippi., Results: Twenty-eight colonies (21 colonies from Mississippi, six from Louisiana, one from China) were collected. Sequencing and analysis of 112 sequences revealed 15 haplotypes of cytochrome oxidase subunit II (COII) gene in the world. Two haplotypes of COII were identified in Mississippi. In addition, specific primers were designed and tested differentially to amplify characteristic fragments for verifying and surveying different genotypes of C. formosanus in the future., Conclusion: Of the two haplotypes identified in Mississippi, the GA group was identical to those reported previously in Georgia, Louisiana, Alabama and other infested states. The second haplotype, the AT group, was identified for the first time in southeastern United States. Sequence identity of the AT-group C. formosanus with those reported mainly in southeastern Asian countries provided evidence of at least two introductions of C. formosanus into the United States.