The natural distribution ofthe honeybee (Apis mellifera L.) hás been changed by humans in recent decades to such an extent that the formerly widest-spread European subspecies, Apís mellifera mellifera, is threatened by extinction through introgression from highly divergent commercial strains in large tracts of its range. Conservation efforts for A. m. mellifera are underway in multiple European countries requiring reliable and cost-efficient molecular tools to identify purebred colonies. Here, we developed four ancestry-informative SNP assays for high sample throughput genotyping using the iPLEX Mass Array system. Our customized assays were tested on DNA from individual and pooled, haploid and diploid honeybee samples extracted from different tissues using a diverse range of protocols. The assays had a high genotyping success rate and yielded accurate genotypes. Performance assessed against whole-genome data showed that individual assays behaved well, although the most accurate introgression estimates were obtained forthe fourassays combined (117 SNPs). The best compromise between accuracy ana genotyping costs was achieved when combining two assays (62 SNPs). We provide a ready-to-use cost-effective tool for accurate molecular identification and estimation of introgression leveis to more effectively monitor and manage A. m. mellífera conservatories.