Simple SummaryThe human management of honey bees (Apis mellifera) has led to a reduction in the range and integrity of native subspecies, as they are replaced by foreign subspecies with traits that are perceived to be more desirable. Introgression—the transfer of alleles (gene variants) between genetically distinct lineages via hybridisation and repeated backcrossing—can lead to the loss of combinations of alleles that have built up over time as a result of adaptation. Measuring introgression is important for assessing the genetic integrity of colonies for breeders and conservationists. However, there is no agreed upon approach for measuring introgression in honey bee colonies. Here, we compare two commonly applied statistical methods for estimating introgression in honey bees from both individual and colony-pooled genetic data: a clustering approach (ADMIXTURE) and an allele pattern approach (ABBA BABA). Overall, data from a single individual from a colony resulted in lower introgression estimates than those from pooled colony samples using ADMIXTURE. However, ABBA BABA consistently yielded lower introgression estimates than ADMIXTURE. This study highlights that sometimes one individual is not enough to assess colony-level introgression and future studies using pooled colony approaches should not be solely dependent on ADMIXTURE for introgression estimates.AbstractThe human management of honey bees (Apis mellifera) has resulted in the widespread introduction of subspecies outside of their native ranges. One well known example of this isApis mellifera mellifera, native to Northern Europe, which has now been significantly introgressed by the introduction of C lineage honey bees. Introgression has consequences for species in terms of future adaptive potential and long-term viability. However, estimating introgression in colony-living haplodiploid species is challenging. Previous studies have estimated introgression using individual workers, individual drones, multiple drones, and pooled workers. Here, we compare introgression estimates via three genetic approaches: SNP array, individual RAD-seq, and pooled colony RAD-seq. We also compare two statistical approaches: a maximum likelihood cluster program (ADMIXTURE) and an incomplete lineage sorting model (ABBA BABA). Overall, individual approaches resulted in lower introgression estimates than pooled colonies when using ADMIXTURE. However, the pooled colony ABBA BABA approach resulted in generally lower introgression estimates than all three ADMIXTURE estimates. These results highlight that sometimes one individual is not enough to assess colony-level introgression, and future studies that do use colony pools should not be solely dependent on clustering programs for introgression estimates.