The allele specificity of AFLP markers was assessed in five relatively unrelated potato genotypes. To this end, two diploid mapping populations of potato, F1SH x RH and F1AM x RH, were analysed using four and six AFLP primer combinations, respectively, recently applied to the analysis of the genetically well characterized backcross population BC_C x E. The AFLP profiles of the five parents revealed 733 AFLP markers and, when identical primer combinations were used, 131 comigrating AFLP markers were identified. After construction of five parental maps, the genomic positions of these comigrating AFLP markers were compared and 117 markers (89%) which targeted the same genomic region were assumed to be homologous. Of these putative homologues, 20 markers, each cloned from at least two genotypes, were sequenced and 19 sets of amplification products were shown to be nearly identical. The number of AFLP markers previously mapped in population BC_C x E ranged from three to eleven per chromosome, which allowed a reliable assessment of chromosome numbers from individual linkage groups obtained in populations F1SH x RH and F1AM x RH. The high incidence of corresponding AFLP alleles was confirmed by using an additional set of five primer combinations. The 733 AFLP markers localized provide a valuable reference collection for future mapping studies in potato. As a consequence AFLP analysis may replace more laborious locus-specific marker techniques.