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Ten simple sequence repeat (SSR) loci were used to study polymorphism in 54 almond genotypes. All genotypes used in this study originated from almond-growing areas in Tunisia with different climatic conditions ranging from the sub-humid to the arid and are preserved in the national collection at Sidi Bouzid. Using ten SSR, 130 alleles and 250 genotypes were revealed. In order to develop an identification key for each accession, the data were analysed separately for each microsatellite marker. The most polymorphic microsatellite (CPDCT042) was used as a first marker. Two microsatellite loci (CPDCT042 and CPDCT025) were sufficient to discriminate among all accessions studied. Neighbour-joining clustering and principal coordinate analysis were performed to arrange the genotypes according to their genetic relationships and origin. The results are discussed in the context of almond collection management, conformity checks, identification of homonyms, and screening of the local almond germplasm. Furthermore, this microsatellite-based key is a first step toward a marker-assisted identification almond database.