Honey bees, Apis mellifera L. are the principle managed pollinator of agriculture and horticulture crops in the United States. Apis mellifera is not native to the United States and the first record of this species in the United States was during the early to mid 17th century when European settlers brought it to the United States. The mitochondrial DNA COI-COII intergenic region of A. mellifera exhibits a high degree of genetic variability within and among A. mellifera lineages and is useful for differentiating lineages as well as detecting unique mitotypes. We conducted a study of the genetic diversity of honey bees from central and south central United States from primarily feral populations. Of the 469 samples from 14 states subjected to DNA sequencing we found evidence of four mitotypes from the 'O' lineage: O5, O5d, O5"b, and O2. Only one of these mitotypes, O2, has been previously observed (in Lebanon). Within the feral population, this lineage accounted for 5% of the observed mitotypes. Of the 24 'O' lineage samples mitotype O5 was the most common and accounted for 52% of the total observed 'O' mitotypes. Bayesian and maximum parsimony (MP) phylogenetic analysis revealed that O2, O5, and O5d were more closely related to those found in Libya (O5b, O5a, O4a, O4b), Lebanon (O16, O2, O3), and Egypt (O1c). However, O5"b appears to have no close relationship to any of the other mitotypes. The existence of the Middle Eastern 'O' lineage in the south central and central United States suggests that further molecular genetic studies of the honey bee population is needed for utilizing and conserving the genetic variation which most likely exists in the Unites States. Furthermore, this study also suggests that feral honey bees are surviving despite the introduction of the varroa mite in the 1980s which reduced the feral and managed populations.