A highly polymorphic locus in the honey bee, Apis mellifera L., was detected with genomic probe pB178. Eighty-five alleles, consisting of MspI and DdeI RFLPs, were found among the Old and New World bees tested. Forty-one MspI and 43 DdeI restriction fragment patterns, or variants, were identified. Variants and alleles were discontinuously distributed in Old World European and African subspecies. Principal coordinate analysis of the genetic distances between the alleles resulted in the identification of three distinct groups corresponding to three groups of honey bee races with historically different geographical distributions: east European A. m. ligustica and A. m. caucasica; west European A. m. mellifera; and South African A. m. scutellata. The clustering of alleles into these groups is consistent with previous honey bee phylogeographic studies, employing other nuclear and mitochondrial DNA markers, which in part support the evolutionary history of the honey bee hypothesized by Ruttner based on morphometric and allozyme data. The majority of alleles in bees from the USA grouped with those found in east European bees, while other alleles grouped with alleles found in A. m. mellifera. While the majority of the alleles in neotropical bees grouped with or were identical to African alleles, other alleles grouped with alleles found in A. m. mellifera, A. m. ligustica, and A. m. caucasica. Clues to the ancestry of neotropical bees may be found in the identification of alleles that were identical or more similar to alleles found in South African and west European bees; evidence for west European ancestry has been suggested using other taxonomic characters that were not unique to west European bees. Both west European and African alleles were found in individual neotropical colonies, which may indicate that honey bee subspecies which evolved allopatrically have hybridized in the human-assisted extension of their original geographical ranges.