Until recently, African and European subspecies of the honeybee (Apis mellifera L.) had been geographically separated for around 10,000 years. However, human-assisted introductions have caused the mixing of large populations of African and European subspecies in South and Central America, permitting an unprecedented opportunity to study a large-scale hybridization event using molecular analyses. We obtained reference populations from Europe, Africa, and South America and used these to provide baseline information for a microsatellite and mitochondrial analysis of the process of Africanization of the bees of the Yucatan Peninsula, Mexico. The genetic structure of the Yucatecan population has changed dramatically over time. The pre-Africanized Yucatecan population (1985) comprised bees that were most similar to samples from southeastern Europe and northern and western Europe. Three years after the arrival of Africanized bees (1989), substantial paternal gene flow had occurred from feral Africanized drones into the resident European population, but maternal gene flow from the invading Africanized population into the local population was negligible. However by 1998, there was a radical shift with both African nuclear alleles (65%) and African-derived mitochondria (61%) dominating the genomes of domestic colonies. We suggest that although European mitochondria may eventually be driven to extinction in the feral population, stable introgression of European nuclear alleles has occurred.