<p>The honey bee <i>Apis mellifera</i> Linnaeus, 1758 has a worldwide distribution with more than 30 subspecies recognized by morphological and genetic methods. Estonia is within the distribution area of the European dark honey bee (<i>A. m. mellifera</i>), although other subspecies have been imported by humans over the years. Our aim was to perform an assessment of the genetic diversity present in Estonian honey bee populations. We analysed the mitochondrial DNA (mtDNA) “COI-COII intergenic region” from 156 honey bees collected from 35 stationary apiaries. We found that the Estonian honey bee population is mostly comprised of two subspecies within the mitochondrial lineage C: <i>A. m.</i> ligustica (55.1%) and <i>A. m.</i> carnica (35.9%), both native subspecies to southern Europe. The Estonian native subspecies (<i>A. m.</i> mellifera; lineage M) was only detected in 7.7% of the samples. A single colony of <i>A. m. iberiensis</i> (native to Iberian Peninsula) was identified. The phylogenetic analyses revealed eleven mtDNA haplotypes organized in three clearly distinct clusters: <i>A. m. mellifera</i>, <i>A. m. iberiensis</i> and <i>A. m. ligustica</i> + <i>A. m. carnica</i>. Overall, the Estonian honey bee population is mainly comprised of commercial C-lineage honey bees, possibly imported from southern Europe. The native <i>A. m. mellifera</i> subspecies is rare, despite representing a potential reservoir of unique combinations of genes adapted to local conditions. The new data may contribute to the implementation of conservation projects and to better understand the hybridization between honey bee subspecies.</p>