The genetic analysis of mitochondrial DNA (mtDNA) provides substantial information for evolutionary studies. A relatively small size compared with nuclear DNA, a rapid rate of sequence divergence, and maternal inheritance, make the mtDNA a useful tool for molecular phylogenetic studies. Here, we investigated and analyzed the mtDNA of five species of honeybees, Apis mellifera, A. cerana, A. dorsata, A. laboriosa and A. florea collected from Japan, Nepal, and India. A 528-bp DNA fragment of the NADH dehydrogenase subunit 4 (ND4 according to A. mellifera's mt DNA sequence) was PCR-amplified and sequenced. The DNA and deduced amino acid sequences were aligned and the phylogenetic relationships were investigated. The results indicate the genetic diversity of this functional protein among different honeybee species. For these studied species, a high A+T content (from 78.21 to 79.74%) was observed. Of the three colon positions, the third and the second positions show the most AT bias (91.93%) and the least AT bias (69.66%), respectively. The extremely high A+T content in the gene leads to high A → T and T → A transversion substitutions and the biases of colon usage. The phylogenetic trees were constructed with maximum likelihood method for the amino acid and the nucleotide sequences of ND4. For the analysis of nucleotide sequences, the first and second positions and the third position of the colons were separately investigated. All trees showed that evolutionary relationship between A. mellifera and A. cerana was close as well as between A. dorsata and A. laboriosa, while the phylogenetic position of A. florea was uncertain.