Two nuclear honeybee DNA probes, 12RlC1 and 2A2, were reported previously to detect restriction fragment patterns specific to African and neotropical African honeybee populations. Individual drones and workers from several additional Old and New World populations, African and European, were tested further with these probes. With probe 12RlC1, only two of several Hhal fragment patterns were seen among haploid drone progeny of each queen bee, indicating that the patterns represented alleles at a single locus. Four alleles detected by probe 12RlC1 were described previously, three of which had been found only in populations of African descent. In this study, one of the three was found at a low frequency among samples from western Europe, northern Mexico, and the United States. However, ten additional alleles were discovered in South African drones, six of which were seen also in neotropical African colonies. With probe 2A2, only one or the other of two Alul restriction fragments was detected in drones, indicating that the fragments represented alleles at a single locus. One of the two alleles, seen previously only in populations of African descent, was found at a very low frequency in bees from western Europe and northern Mexico.