At the present time, the uncontrolled interbreeding of bees that belong to different subspecies or races of Apis mellifera has become a threat in Ukraine. Conserving the gene pool of local bee breeds, which are well adapted to the local environmental conditions, is an urgent problem that cannot be solved without the application of molecular methods for monitoring of the genetic composition of local populations. To clarify the origin of the Carpathian bee and to develop molecular methods for its identification, sequencing and comparative analysis of the CoI–CoII region of the mitochondrial genome of the Carpathian bee and representatives of several subspecies of A. mellifera was performed. In the studied region, a mutation specific for the Carpathian bee was discovered. It was found that Carpathian bee originates from the Central European subspecies A. m. carnica and represents the ecotype that arose from the migration of this subspecies to the East, which resulted in the formation of Carpathian populations of honey bees.