AbstractAlthough much research has been conducted to characterize microsatellites and develop markers, the distribution of microsatellites remains ambiguous and the use of microsatellite markers in genomic studies and marker-assisted selection is limited. To identify microsatellites for cotton research, we mined 100,290, 83,160 and 56,937 microsatellites with frequencies of 41.2, 49.1 and 74.8 microsatellites per Mb in the recently sequencedGossypiumspecies:G. hirsutum,G. arboreum,andG. raimondii,respectively. The distributions of microsatellites in their genomes were non-random and were positively and negatively correlated with genes and transposable elements, respectively. Of the 77,996 developed microsatellite markers, 65,498 were physically anchored to the 26 chromosomes ofG. hirsutumwith an average marker density of 34 markers per Mb. We confirmed 67,880 (87%) universal and 7,705 (9.9%) new genic microsatellite markers. The polymorphism was estimated in above three species byin silicoPCR and validated with 505 markers inG. hirsutum. We further predicted 8,825 polymorphic microsatellite markers withinG. hirsutumacc. TM-1 andG. barbadensecv. Hai7124. In our study, genome-wide mining and characterization of microsatellites and marker development were very useful for the saturation of the allotetraploid genetic linkage map, genome evolution studies and comparative genome mapping.