Microsatellite markers, also known as SSRs, comprise a keystone technology for genetic linkage analysis, QTL mapping, marker-assisted breeding, and genome analysis. In order to contribute to a growing body of molecular marker resources for cotton research and improvement, 700 primer pairs were developed to amplify new microsatellite markers, designated Gh for Gossypium hirsutum. These primers were designed using microsatellite sequences that were isolated from genomic DNA of G. hirsutum cv. Tamcot Sphinx by a biotinylated-oligonucleotide capture method. A total of 4,512 clones from (GA)n, (AGA)n, and (CA)n microsatellite-enriched libraries were sequenced. From these, 1,059 primer pairs were developed. Of the first 700 primer-pairs to be characterized, 602 primer pairs (86%) produced one or more distinct PCR amplification products within the expected size range in at least one of the test cotton genotypes, G. hirsutum acc. TM-1 and G. barbadense acc. 3-79. Further, 201 primer pairs (28.7%) yielded size polymorphisms between TM-1 and 3-79 that were easily resolved using high-resolution agarose electrophoresis. A subset of 165 polymorphic markers was fully genotyped on a TM-1 x 3-79 interspecific recombinant inbred (RI) population of 191 individuals. In this analysis, segregation distortion was low (10.3% of loci) and functional redundancy of marker loci was low (1.2% of loci). Data from these markers are being incorporated in an integrated SSR map from the TM-1 x 3-79 recombinant inbred population. Updates regarding sequence, primer, polymorphism, and linkage information from the remainder of the Gh microsatellite collection will be uploaded directly to CottonDB and CMD databases.