The genetic diversities of 50 upland cotton cultivars, which had been and I ro are being popularized in Xinjiang province in recent 50 years, were investigated hased on EST-SSR markers. Based on the 42 EST-SSRs, a total of 81 polymorphism loci had been detected in these cultivars. For each SSR primer pair, 1 to 3 polymorphisms with an average of 2.4 could be detected. Based on EST-SSR data, the coefficient of similarity among 50 upland cotton cultivars was calculated varying from 0.11 to 0.83. It was indicated that more genetic diversities among those cultivars were presented, while genetic diversity in same origination in Xinjiang had been narrowed. Cluster analysis revealed that these 50 cultivars can be divided into two major subgroups based on the coefficient of similarity, and most of the tested cultivars were consistent with Xinjiang's cotton lines in pedigree, containing the constituent of the former Soviet Union germplasms. It was testified that EST-SSR markers derived directly in transcribed regions of cotton cultivars.