Africanized honeybees, hybrids between African (Apis mellifera scutellata) and western European (A. m. mellifera) and eastern European (A. m. caucasica, A. m. carnica, and A. m. ligustica) subspecies are widely distributed in urban areas of the southwestern USA. However, little is known about their distribution in rural regions. We collected bees at 54 sites in a 5,350-km² study area in the Sonoran Desert of southwestern Arizona. We used mitochondrial DNA (mtDNA) analysis of individual worker honeybees (10 per site) to assess genetics of colonies within the study area. Among collected bees, 86.9% possessed African mtDNA. Western European, eastern European, and Egyptian (A. m. lamarckii) mtDNA was present in 5.6%, 4.1%, and 3.4% of collected bees, respectively. There was no apparent relationship between the percentage of bees having African mtDNA and distance to agricultural fields or elevation of the collection site. The preponderance of Africanized honeybees confirms previous studies and predictions concerning their distribution in the southwestern USA.