Mitochondrial DNA sequence variation can be used to infer honey bee evolutionary relationships. In this study, DNA sequence diversity in the ND5 region of the mitochondrial genome was investigated in 93 samples of Apis mellifera from 15 different populations in Turkey. Five novel haplotypes were revealed for the ND5 gene segment of Turkish honeybees. The number of variable sites found was 6 for this region while 2 were parsimony informative sites. The average pairwise genetic distances were 0.3% for ND5 gene. In this study, the NJ tree of ND5 gene segment were constructed with the published sequences of Apis mellifera haplotypes. This study expands the knowledge about the mitochondrial ND5 region in Apis mellifera and it is also the first comprehensive sequencing analysis of ND5 region in Turkish honeybees.