Background Sweet-seeded domesticated almonds were brought to the Mediterranean Basin from central Asia about 4000 years ago. In Italy, most of the almonds produced are cultivated in the southern part of the country. Local populations of the tree in Sardinia are largely seed-derived and mostly self-incompatible, so have developed extensive genetic diversity. The need to protect biodiversity has prompted a revived interest in local genetic materials in almond. Two Italian collections have been established, one in Sardinia and the other in Apulia. These collections were the focus of the present evaluation of genetic diversity. Results Eleven SSRs (microsatellites) were used for fingerprinting. The Sardinian germplasm was highly polymorphic, revealing a mean of 14.5 alleles per locus and a mean heterozygosity of 0.71. Using a model-based clustering approach, two genetic clusters were distinguished: one included all the commercial varieties and most of the Sardinian accessions, and the other most of the Apulian accessions. A similar structure was produced using a distance-based cluster analysis. The Sardinian accessions could still be distinguished from the commercial germplasm with few exceptions. Conclusion The extensive genetic variability present in the Sardinian and Apulian almond germplasm indicates that these materials represent an important source of genes for the improvement of the crop.