Maintenance of redundant garlic (Allium sativum L.) accessions is expensive due to the necessity of yearly regenerating garlic accessions in germplasm centers. Therefore, rapid characterization of garlic accessions is important for avoiding duplicated genotypes. For this purpose we developed several locus-specific polymerase chain reaction (PCR)-based DNA markers, and tested them for the characterization of garlic clones that were previously analyzed using amplified fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) markers. Among 25 garlic clones, 4 locus-specific markers generated 24 polymorphic alleles. Garlic accessions within a group determined by AFLP and RAPD analyses generally had the same alleles as the locus-specific markers. In addition. several alleles of these locus-specific markers were associated only with certain AFLP and RAPID groups, which can allow rapid assessment of newly collected garlic accessions; therefore. these locus-specific markers can be used as another tool for the rapid characterization of garlic germplasm collections.