Simple sequence repeats(SSRs) have been widely applied as molecular markers in genetic studies.However,the number of expressed sequence tags(ESTs) and SSR markers from Gossypium barbadense is fewer than those from other cotton species.In this study,EST-SSR distribution from G.barbadense was characterized and new G.barbadense-derived EST-SSR markers were determined on the basis of the ESTs obtained by randomly sequencing 2 cDNA libraries associated with fiber development in G.barbadense.By mining 9697 non-redundant ESTs,a total of 638 SSR loci derived from 595 ESTs were observed.In G.barbadense,the frequency of ESTs containing SSRs was 6.13%,with an average of 1 SSR in every 10.4 kb of EST sequence.Furthermore,trinucleotide was found to be the most abundant repeat type among 2-6-nucleotide repeat types.It accounted for 26.6% of the total,followed by the hexanucleotide(26.0%) and pentanucleotide repeats(25.9%).Among all the repeat motifs,(AAG)n accounted for the highest proportion.EST-SSR primer pairs were developed using the Primer3 program,and the redundant primers were removed using the virtual PCR approach.As a result,380 non-redundant EST-SSR primer pairs were developed and used to detect polymorphisms between the mapping parents G.hirsutum ’TM-1’ and G.barbadense ’Hai7124’ for constructing linkage groups in cultivated allotetraploid cotton.Out of these,98(25.8%) primer pairs detected polymorphisms.Finally,95 polymorphic loci from 82 primer pairs were integrated into the backbone genetic map;of these,42 were mapped into the A subgenome and 53 into the D subgenome.The present work provided the foundation for constructing saturated genetic maps and conducting comparative genomic studies on different cotton species. Simple sequence repeats (SSRs) have been widely applied as molecular markers in genetic studies. However, the number of expressed sequence tags (ESTs) and SSR markers from Gossypium barbadense is fewer than those from other cotton species. In this study, EST-SSR distribution from G. barbadense was characterized and new G. barbadense-derived EST-SSR markers were determined on the basis of the ESTs obtained by randomly sequencing 2 cDNA libraries associated with fiber development in G. barbadense. BY mining 9697 non-redundant ESTs, a total of 638 SSR loci derived from 595 ESTs were observed. In G. barbadense, the frequency of ESTs containing SSRs was 6.13%, with an average of 1 SSR in every 10.4 kb of EST sequence. Futuremore, trinucleotide was found to be the most abundant repeat type among 2-6-nucleotide repeat types. Intreated for 26.6% of the total, followed by the hexanucleotide (26.0%) and pentanucleotide repeats (25.9%). Among all the repeat motifs, (AAG) n accounted for the highest proportion.EST-SS R primer pairs were developed using the Primer3 program, and the redundant primers were removed using the virtual PCR approach. As a result, 380 non-redundant EST-SSR primer pairs were developed and used to detect polymorphisms between the mapping parents G.hirsutum ’ TM-1 ’and G. barbadense’ Hai7124 ’for constructing linkage groups in cultivated allotetraploid cotton. Out of these, 98 (25.8%) primer pairs detected polymorphisms. Finaally, 95 polymorphic loci from 82 primer pairs were integrated into the backbone genetic map ; of these, 42 were mapped into the A subgenome and 53 into the D subgenome. The present work provided the foundation for constructing saturated genetic maps and conducting a comparative genomic study on different cotton species.