Genotype and allele frequencies of the DYS19, D12S67, and D1S80 highly polymorphic loci were determined in population samples from southern Italy (103 subjects) and Greece (84 subjects) using the amplified fragment length polymorphism (AFLP) technique (polymerase chain reaction followed by native PAGE and silver staining). Five, eleven, and eighteen alleles were found at the DYS19, D12S67, and D1S80 loci, respectively. PIC values ranged from 0.55 (DYS19 locus in Italians) to 0.79 (D12S67 locus in Italians). The distribution of D12S67 and D1S80 genotypes conformed to Hardy-Weinberg equilibrium, as confirmed by three statistics. Heterogeneity G tests, carried out on allele frequency distributions, showed a significant difference between the samples at the DYS19 locus, whereas no difference was found with regard to the other polymorphisms. Using data from the literature, we widened the comparison to other European groups analyzed for the same markers. All the polymorphisms were found to distinguish between populations of the same main ethnic group. In particular, D1S80 allele frequencies distinguished the Finns from other European groups (Spanish, German, Italian, and Greek samples). The reduced assay time, the high polymorphism level, and the ability to distinguish between populations indicate that these markers have potential value in population genetic studies.