Honey bee, Apis mellifera L. (Hymenoptera: Apidae) DNA has alleles, at three polymorphic restriction sites, which were initially found almost exclusively in samples from eastern Europe. These markers were first detected with three labeled, cloned, probes on DNA blots. In this study, the polymorphic sites were made easier to analyze with the polymerase chain reaction (PCR). The amplification primers and conditions are described. The regions with the polymorphic sites were mapped within the sequences of the honey bee genome and were found within three separate linkage groups. The genomic locations of the defining restriction sites and PCR primers are given. With the PCR-based method of detection, additional samples of bees from Europe and Africa were examined. Within the eastern European group, the three markers were at high frequencies in Italian bees (subspecies Apis mellifera ligustica Spinola), two markers were at high frequencies and one marker was at a lower frequency in Carniolan bees (Apis mellifera carnica Pollman), and all three markers were absent or at low frequencies in Caucasian bees (Apis mellifera caucasica Gorbachev). The three markers were absent or at low frequencies in western European bees (Apis mellifera mellifera L and Apis mellifera iberica Goetze) and in South African bees (Apis mellifera scutellata Lepeletier).