Since the rapid spread and establishment of Africanized honey bee populations in South America, Africanized bees have persisted as the dominant strain. Remarkably, Chile has remained free of Africanized bee populations, making the country a valuable exporter of mated queens. Given Chile's pivotal role in the apiculture industry, monitoring the genetic makeup of its honey bee colonies is crucial, yet documentation has been limited to a few studies. Here, we evaluate the ancestral composition of honey bees across eleven different regions in Chile. We find that Chilean honey bees have low levels of admixture, which is markedly lower relative to commercial colonies located internationally. The genetic ancestry of Chilean honey bees is primarily of Eastern European origin, with low levels of Western European ancestry. Finally, we detect a significant relationship between geography and genetic ancestry, suggesting regional adaptations that warrant further investigation.