We have used SSRs derived from both genomic and cDNA clones, and the self-incompatibility alleles to analyse the almond germplasm in China. Almond cultivars from other countries were also included in the analyses. Fifty-four SSR markers were developed and 27 were used for the phylogenetic analysis and fingerprinting. A phylogenetic tree was constructed based on the 178 polymorphic alleles distinguished among the 45 accessions. Almond cultivars from China and the Mediterranean region were classified in two independent groups. Thirty-six new S-RNase gene sequences have been amplified by PCR and sequenced from the 28 almond cultivars and other almond species of China. The S-genotypes have been identified in some of the cultivars. Phylogenetic analysis suggested that the S-genes have different evolutionary history after almond was introduced from the Central and Western Asia to the Mediterranean region, which might have been related to their geographic origin.