Mitochondrial DNA sequence variation can be used to infer honeybee evolutionary relationships. In this study, DNA sequence diversity of the mitochondrial 16s rDNA region was investigated in 112 honeybees from 15 populations in Turkey, which is mainly populated with Apis mellifera anatoliaca, A. m. caucasica, and A. m. meda. The study revealed 11 haplotypes for this segment, with 13 variable sites and nine parsimony informative sites. The haplotypes were not discriminated according to their geographical locations in a neighbor-joining dendrogram based on 16s rDNA sequences available in Genbank, but all the haplotypes obtained in this study are clustered with published haplotypes such as A. mellifera TAS (AF214666) and A. m. ligustica (EF116868) and with some unpublished Genbank records (HQ318928, HQ318934, and HQ318938). This study expands the knowledge of the mitochondrial 16s rDNA region, and it presents the first comprehensive sequence analysis of this region in Turkish honeybees.