A polymerase chain reaction (PCR)-based restriction fragment-length polymorphism (RFLP) assay was developed that discriminates among the 4 mitotypes found in North, Central, and South American honey bee racial groups—eastern European (Apis mellifera ligustica Spinola, caucasica Gorbachev, and carnica Pollman), western European (A. m. mellifera Linnaeus), Egyptian (A. m. lamarckii Cockerell Lepeletier), and sub-Saharan African (A. m. scutellata). Before the development of this assay, 13% of southern Californian feral bees collected before the arrival of the Apis mellifera scutellata (Africanized) race were found to contain a non-European mitochondrial genotype that could not be distinguished from that of A. m. scutellata. DNA sequence analysis suggests the unusual mitotype to be that of A. m. lamarckii. An RFLP polymorphism was identified that distinguished this subspecies from all others present in North America. This polymorphism was not found in any of 96 bees collected primarily in Mexico and Central America. Thus, the Egyptian mitochondrial type is either absent or extremely rare in these regions. The PCR assay also distinguishes A. m. lamarckii from 2 other north African racial types, A. m. intermissa Buttel-Reepen and A. m. sahariensis Baldensperger.