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The present study aimed to characterize the simple sequence repeat markers in cotton using the cotton expressed sequence tags. A total of 111 EST-SSR polymorphic molecular markers with trinucleotide motifs were used to evaluate the 79 accessions of Gossypium L., (G. darwinii, 59 and G. barbadense, 20) collected from the Galapagos Islands. The allele number ranged from one to seven, with an average value of 2.85 alleles per locus, while polymorphisminformation content values varied from 0.008 to 0.995, with an average of 0.520. The discrimination power ranks high for the majority of the SSRs, with an average value of 0.98. Among 111 pairs of EST-SSRs and gSSRs, a total of 49markers, comprising nine DPLs, one each of MonCGR, MUCS0064, and NAU1028, and 37 SWUs (D-genome), were found to be the best matched hits, similar to the 155 genes identified by BLASTx in the reference genome of G. barbadense, G. arboreum L., and G. raimondii Ulbr. Related genes GOBAR_DD21902, GOBAR_DD15579, GOBAR_DD27526, and GOBAR_AA04676 revealed highly significant expression 10, 15, 18, 21, and 28 days post-anthesis of fiber development. The identified EST-SSR and gSSR markers can be effectively used for mapping functional genes of segregating cotton populations, QTL identification, and marker-assisted selection in cotton breeding programs. [ABSTRACT FROM AUTHOR]