Abstract Background Eusocial insects play a central role in many ecosystems, and particularly the important pollinator honeybee ( Apis mellifera ). One approach to facilitate their study in molecular genetics, is to consider whole colonies as single individuals by combining DNA of multiple individuals in a single pool sequencing experiment. Such a technique comes with the drawback of producing data requiring dedicated analytical methods to be fully exploited. Despite this limitation, pool sequencing data has been shown to be informative and cost-effective when working on random mating populations. Here, we present new statistical methods for exploiting pool sequencing data of eusocial colonies in order to reconstruct the genotype of the colony founder, the queen. This leverages the possibility to monitor genetic diversity, perform genomic-based studies or implement selective breeding. Results Using simulations and honeybee real data, we show that the methods allow for a fast and accurate estimation of the genetic ancestry, with correlations of 0.9 with that obtained from individual genotyping, and for an accurate reconstruction of the queen genotype, with 2% genotyping error. We further validate the inference using experimental data on colonies with both pool sequencing and individual genotyping of drones. Conclusion In this study we present statistical models to accurately estimate the genetic ancestry and reconstruct the genotype of the queen from pool sequencing data from workers of an eusocial colony. Such information allows to exploit pool sequencing for traditional population genetics, association studies and selective breeding. While validated in Apis mellifera , these methods are applicable to other eusocial hymenoptera species.