Simple sequence repeats (SSR) markers have greater advantages relative to other molecular markers in genetic analysis due to their Mendelian inheritance and co-dominance expression features. Numerous expressed sequence tags (EST) of genus Casuarina, now available from public databases, make it possible to develop EST-SSR markers with high efficiency at low cost. From the analysis of 12,063 EST derived from casuarinas, 367 SSR loci were identified from 352 unigenes, and the distribution frequency of SSR loci was 3.0%. The di-and trinucleotide accounted for 57.77 and 34.06% respectively. They comprised overwhelming majority in five types of repeat motifs of SSR, and AG/CT was the most predominant motif type of SSR. A total of 79 primer pairs were designed based on the non-redundant EST-SSR sequences, but only 21 were successfully amplified and generated polymorphic SSR products in four different Casuarina species and eight clones of C. equisetifolia. Eight primer pairs out of the 21 were applied for paternity analyses of open-pollinated progenies of one female individual of C. equisetifolia. At 95% confidence interval, 76 male individuals were identified as pollen parents of 262 out of 461 progenies, but at 80% confidence interval, 84 male individuals were identified as father trees of 328 out of the 461 progenies.