We carried out a study which involved DNA sequencing of a portion of the mitochondrial DNA COI-COII region of Africanized honey bees (AHB) from the USA. A total of 12 mitotypes were observed, of which seven have not been previously described. Of the 172 samples, two mitotypes, A1 and A1d, accounted for 77% of the observed mitotypes, while mitotypes A1a, A26c, A26d, A29a, and A30 were only observed once. A possible reason why these new mitotypes have not been described before is because previous studies on AHB in the new world have relied primarily on PCR-restriction fragment length polymorphism (RFLP), which is less sensitive than DNA sequence data. Multiple mitotypes of ‘A’ lineage honey bees have previously been observed in South America and Mexico using PCR-RFLP and DNA sequence analysis. Our findings are consistent with previous studies of AHB genetic variation from central Mexico, Columbia, and northern Brazil, in that the A1 mitotype was more common than the A4. Maximum parsimony analysis revealed that all of the ‘A’ lineage Apis mellifera mitotypes formed a distinct clade relative to representatives of the ‘M’, ‘C’, and ‘O’ lineages. Statistical analysis of the mitotype frequencies in the USA revealed an excess of low frequency mitotypes, indicating that the population size is expanding. The amount of genetic variation observed in Africanized honey bees in the USA therefore supports the idea that there have been multiple introductions of AHB into the country.