Microsatellite [simple-sequence repeat (SSR)] markers were developed and positioned on the genetic map of tetraploid cotton. Three hundred and ninety-two unique microsatellite sequences, all but two containing a (CA/GT) repeat, were isolated, and the deduced primers were used to screen for polymorphism between the Gossypium hirsutum and G. barbadense parents of the mapping population analyzed in our laboratory. The observed rate of polymorphism was 56%. The 204 polymorphic SSRs revealed 261 segregating bands, which ultimately gave rise to 233 mapped loci. The updated status of our genetic map is now of 1,160 loci and 5,519 cM, with an average distance between two loci of 4.8 cM. The presence of a total of 466 microsatellite loci, with an average distance of 12 cM between two SSR loci, now provides wide coverage of the genome of tetraploid cotton and thus represents a powerful means for the production of a consensus map and for the effective tracking of QTLs.