The honeybee (Apis mellifera L.) has a large number of geographic subspecies distributed across Europe, Africa and Asia, many of which have been described. This identification is important for bee breeding and preserving honeybee biodiversity. To investigate the origin of Jordanian honeybees, 32 samples collected from different locations in Jordan were analyzed using four different enzyme systems: Bg/II site in cytochrome oxidase b (Cytb), EcoRI site in large ribosomal (lsRNA) subunit, XbaI site in cytochrome c oxidase I (COI) subunit and HinCII site in cytochrome c oxidase I (COI) subunit. The first three enzymes were found to be polymorphic. The DNA banding pattern analyses revealed that Jordanian honeybees belong to the East Mediterranean and Middle Eastern mitochondrial lineages.