The genus Spartina represents a monophyletic lineage of subfamily Chloridoideae, where no member is known to be diploid and polyploidy has played an important role in speciation. Simple sequence repeat (SSR) markers have been developed from a set of expressed sequence tags (ESTs) generated from salt-stressed Spartina alterniflora. Di- and tri- nucleotide repeats were most abundant. Out of 100 SSRs with 5 or more repeats, successful amplification was obtained in 81 cases in an initial screening with 8 S. alterniflora genotypes and 18 SSRs were polymorphic. Fifteen SSRs were studied in detail to study the polymorphism and cross transferability among 6 different species of Spartina; (S. alterniflora, S. cynosuroides, S. spartinae, S. foliosa, S. pectinata, S. patens). Twelve primers showed amplification across all the species, while 100% cross transferability was observed with at least one species. This suggested a high level of conservation of sequences in the transcribed region. Sequence analysis of a SSR locus coding for reversibly glycosylated polypeptide showed stretches of conserved sequence across different species of Spartina and the length polymorphism among the different species was mainly due to the number of tandem repeats within the coding regions. However, single nucleotide polymorphisms (SNPs) were also observed in the sequences in the coding regions of different species outside the repeat region. A single locus sequence-based phylogeny grouped S. alterniflora and S. foliosa in one group. The same was the result with the tree formed with 10 SSRs spanning 56 loci where S. alterniflora clubbed with S. foliosa in one clad that also included S. spartinae and S. pectinata. The second clad consisted of S. cynosuroides and S. patens. Our study shows that the EST-derived SSR markers might be more useful in genetic diversity, evolutionary and comparative genomics studies in Spartina as opposed to genomic SSRs.