Apis mellifera scutellata was introduced to Brazil in 1956 and Africanized honeybee populations have now spread from Argentina to the southwestern United States. Temperate climatic restrictions seem to be a natural limit to Africanized honeybee expansion around parallels 35° to 40° SL. We used allozyme loci (Mdh-1 and Hk-1) and mtDNA haplotypes to characterize honeybee populations in southern Brazil and Uruguay and define a possible transition area between Africanized and European bees. Samples of 194 bee colonies were collected from ten localities between 30°-35° SL and 52°-59° WL. The mtDNA restriction patterns of these colonies were obtained through digestion of the mitochondrial genome by Eco RI, or by digestion by Bgl II and Xba I of the cytochrome B locus and the COI-COII intergenic region, respectively. The distribution limit of African bee colonies, i.e., those populations with only the African mtDNA haplotype and with a high proportion of African genes as shown by allozyme analysis, is located in northern Uruguay, with a hybridization zone located farther south in Uruguay. A gradual cline from north to south was observed, confirmed by mtDNA, racial admixture, and genetic distance analyses. No evidence of either gametic disequilibrium between nuclear markers or cytonuclear disequilibrium among the nuclear and mtDNA genotypes was detected, suggesting that the hybridization process has been completed.