Linkage mapping and genetic diversity studies with DNA markers in plant species assume that comigrating bands are identical, or at least that they have homologous sequences. To test this assumption in a plant with a large genome, sequence identities of 7 polymorphic amplified fragment length polymorphism (AFLP) markers of garlic, previously used to estimate similarity in genetic diversity studies, were characterized. Among 37 diverse garlic clones, 87 bands from these 7 polymorphisms were excised, amplicons were cloned, and 2 to 6 colonies were sequenced from each band, to yield a total of 191 DNA amplicons. Of these 87 bands, 83 bands (95.4%) contained AFLP amplicons that were identical or highly homologous to the typical marker of that band; only 4 bands contained amplicons with little homology to the same-sized amplicons of other garlic clones. Of these 83 bands, 64 (73.6%) contained only highly homologous amplicons (> 90% sequence identity), whereas 19 (21.8%) contained both homologous and nonhomologous amplicons, with sequence identities less than 60%. Of the 37 nonhomologous amplicons identified, 25 (67.5%) differed in length from other amplicons in the band. Sequence conservation of AFLP amplicons followed patterns similar to phylogenetic relationships among garlic clones, making them useful for developing simple PCR-based markers in genetic mapping and diversity assessment.