Canadian honey bees, like all honey bees in the New World, originated from centuries of importation of predominately European subspecies, but their precise genetic ancestry has not been investigated. We used a citizen science approach that engaged a diverse group of beekeepers to undertake the largest population genetic study of Canadian honey bees. We used the dataset to characterize the ancestry of Canadian honey bee populations, test if Northern Canadian colonies have a greater proportion of ancestry from subspecies native to Northern Europe, and determine the effectiveness of using single nucleotide polymorphism (SNPs) to distinguish between Canadian bees and the aggressive and invasive Africanized honey bee found from South America to the Southern United States. We genotyped 855 worker honey bees at 91 ancestrally informative SNPs and found very low levels of genetic differentiation within Canada at these SNPs and small but significant differences in ancestry between provinces. Honey bee populations in Northern and Western Canada were more closely related to subspecies from Southern and Mediterranean Europe. We attributed this pattern to differences in importation practices within Canada. Finally, we were able to accurately discriminate between Africanized bees and Canadian bees using the ancestrally informative SNPs, supporting the use of SNPs for accurately detecting Africanized honey bees and providing valuable insights into the genetic structure of Canadian bees, all while engaging beekeepers in the scientific process.