Frequencies are reported for restriction fragment-length polymorphisms (RFLPs) at a highly polymorphic nuclear locus in Old and New World honey bee populations. The distribution of these (RFLPs) alleles (composed of MspI and DdeI variants) had been found previously to be discontinuous among groups of Old World honey bee subspecies, which included A. mellifera mellifera L. (west European), A. m. ligustica Spinola, A. m. caucasica Gorbachev (east European), and A. m. scutellata Lepeletier (African). In this study, ancestry in New World bees was inferred from allele identities and frequencies at this locus in combination with mitochondrial DNA types. In bees from the United States, collected before the invasion of African bees, east and west European alleles were found at frequencies of 83 and 17%, respectively, which is consistent with previously identified nuclear and mitochondrial DNA markers. Colonies from two neotropical countries, Mexico and Honduras, had African mitochondrial DNA and high frequencies of African nuclear DNA alleles. Consistent with previous findings, east European alleles were absent or detected at low frequencies in these colonies. However, west European alleles were found at frequencies from 26 to 31%. These results suggest that queen offspring of the African queens first introduced into Brazil mated with west European drones, incorporating neutral markers that have since remained in the expanding population of feral African bees. The results point to little paternal introgression from managed east European colonies encountered by the African bees spreading through the neotropics.