Runs of homozygosity (ROH) are continuous homozygous segments that arise through the transmission of haplotypes that are identical by descent. The length and distribution of ROH segments provide insights into the genetic
diversity of populations and can be associated with selection signatures. Here, we analyzed reconstructed whole-genome queen genotypes, from a pool-seq data experiment including 265 Western honeybee colonies from Apis mellifera mellifera and Apis mellifera carnica. Integrating individual ROH patterns and admixture levels in a dynamic population network visualization allowed us to ascertain major differences between the two subspecies. Within A. m. mellifera, we identified well-defined substructures according to the genetic origin of the queens. Despite the current applied conservation efforts, we pinpointed 79 admixed queens. Genomic inbreeding (FROH) strongly varied within and between the identified subpopulations. Conserved A. m. mellifera from Switzerland had the highest mean FROH (3.39%), while queens originating from a conservation area in France, which were also highly admixed, showed significantly lower FROH (0.45%). The majority of A. m. carnica queens were also highly admixed, except 12 purebred queens with a mean FROH of 2.33%. Within the breed-specific ROH islands, we identified 14 coding genes for A. m. mellifera and five for A. m. carnica, respectively. Local adaption of A. m. mellifera could be suggested by the identification of genes involved in the response to ultraviolet light (Crh-BP, Uvop) and body size (Hex70a, Hex70b), while the A. m. carnica specific genes Cpr3 and Cpr4 are most likely associated with the lighter striping pattern, a morphological phenotype expected in this subspecies. We demonstrated that queen genotypes derived from pooled workers are useful tool to unravel the population dynamics in A. mellifera and provide fundamental information to conserve native honey bees.