The honey bee Apis mellifera is of major importance for the world’s agriculture and is also suitable for environmental monitoring. It includes several recognized subspecies distinguished by using morphological and morphometric variants. Here, 200 adult worker Apis mellifera honey bees were collected from Hail region, Saudi Arabia. Mitochondrial 16S rDNA was conducted to detect molecular polymorphism among honey bee A. mellifera subspecies. The amplified and sequenced gene regions of mtDNA revealed the presence of two different subspecies of Apis mellifera carnica (gb| MH939276.1) and Apis mellifera jementica (gb| MH939277.1). The sequences were compared with each other and with others retrieved from the GenBank demonstrating a high degree of similarity (up to 72%). The NJ tree indicated that all Apis species are clustered together in one clade in addition to the genetically origin of Apis species within family Apidae as a paraphyletic group within the African lineage.