Recent advances in genomic technologies have generated a large number of expressed sequence tags (ESTs) in cotton. Many of these ESTs are available in public databases, which offer an opportunity to identify simple sequence repeats (SSR) in ESTs by data mining. These sequences may provide an estimate of diversity in the expressed portion of the genome and may be useful for comparative mapping, for tagging important traits of interest, and for additional map-based cloning of important genes. One hundred and thirty-three SSR-containing ESTs (EST-SSRs) were identified by analyzing 9,948 sequences belonging to Gossypium hirsutum L. in GenBank. The EST-SSR sequences and the related EST sequences without SSRs were clustered to reduce redundancy and to develop consensus sequences. Primers were designed for 84 of these EST-SSRs and were tested for their ability to amplify and detect diversity among three lines of G. hirsutum and one line of G. barbadense L. An average of three amplicons was obtained per primer pair. The intraspecies polymorphism rate among the G. hirsutum cotton cultivars was 26% and interspecific polymorphism between G. hirsutum and G. barbadense was 52%. The presence of SSRs in the EST-SSR markers was confirmed by cloning and sequencing of the amplified products of randomly selected primer pairs from four different lines. To explore the potential use of the EST-SSR loci for comparative mapping, these sequences were compared against different plant species using BLAST assuming an e-value of 1E-10 or less as a significant sequence similarity. About 74% of the EST-SSRs were from fiber-related tissues in G. hirsutum, whereas 26% were from other tissues, such as cotton bolls and cottonseed. Fifty-five percent of these EST-SSR sequences matched sequences in G. arboretum L., and 19% of the sequences showed considerable sequence similarity with sequences in the Arabidopsis thaliana (L.) Heynh. genome. In this manuscript, information about the primer sequence, repeat motif, and the degree of polymorphism of cotton EST-SSR markers is reported. A cost-effective strategy to develop EST-SSR markers by exploiting EST databases is demonstrated for the first time in cotton.