Runs of homozygosity (ROH) are continuous homozygous segments that arise through the transmission of haplotypes that are identical by descent (IBD). The length and distribution of ROH segments provide insight into the genetic diversity of populations and are useful to detect selection signatures. Here, we analysed pooled whole-genome sequencing data from 265 Western honey bee colonies from the two subspecies Apis mellifera mellifera and Apis mellifera carnica. Integrating individual ROH patterns and admixture levels in a high-resolution 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 colonies and a fair amount of admixed colonies, despite the current applied conservation efforts. In contrast, A. m. carnica colonies were more inbred and could not be differentiated according to the geographical origin. We identified 29 coding genes in overlapping ROH segments within the two subspecies. Genes embedded in A. m. carnica specific homozygosity islands suggested a strong selection for production and behavioural traits, whilst the identified cuticula protein-coding genes (CPR3 and CPR4) were associated with their breed-specific stripe pattern. Local adaption of the two subspecies could be confirmed by the identification of two genes involved in the response to ultraviolet (UV) light. We demonstrated that colony genotypes derived from pooled honey bee workers are reliable to unravel the population dynamics in A. mellifera and provide fundamental information to conserve native honey bees.