The hive beetle Aethina tumida Murray is a new pest of honey bee colonies in North America. Specimens of A. tumida were collected throughout its current range in the southeastern United States, and from several sites in South Africa. A 1018-bp section of the mitochondrial cytochrome c oxidase I gene was amplified and sequenced in 26 beetles collected from Florida, Georgia, South Carolina, and North Carolina, and in 14 beetles collected from seven sites in South Africa. Mitochondrial DNA variation between all A. tumida samples was <0.8%, which was still considered within the range expected for a single species. The U.S. samples showed two distinct haplotypes, differing by 6 bp (0.6%). Both haplotypes were found across and within several geographic regions, a result consistent with a single introduction into the United States. However, a broad survey of 151 beetles from their new range revealed significant heterogeneity in haplotype frequencies, perhaps resulting from multiple introductions. Although the data do not allow a precise estimate of the point from which A. tumida were accidentally exported from Africa, the close genetic similarity between beetles from the United States and South Africa indicates that studies conducted on beetle physiology, parasites, and pathogens in South Africa will have a direct bearing on populations now found in the United States.