Cotton (Gossypium L.) is a major source of natural fibre for the world's textile industry and is also an important oil plant. Cotton fibre is the main source of textiles, the seeds are used for oil and the rest of the bagasse is a high-protein animal feed. In addition, more than 50 industries use cotton's so-called short fibre. Breeding studies in cotton are generally based on crosses between the best genotypes in terms of yield and fibre quality. However, the narrow genetic diversity of cultivars has a negative impact on cotton breeding programmes. For this reason, the identification of genetic resources and the disclosure of genetic diversity are important. Herein, high-resolution capillary gel electrophoresis was used to determine the genetic diversity of G. hirsutum and G. barbadense genotypes. Using 19 EST-SSR markers, a total of 47 genotypes were screened. The PIC values of the markers used ranged from 0.268 to 0.889. The mean PIC value was calculated to be 0.603. Concerning the clustering, PCoA and population structure analyses gave similar results, and the genotypes could be divided into 3 main groups. Genetic admixture with G. hirsutum was found in some genotypes of G. barbadense species. We can conclude that i) the EST-SSR markers used in this study are effective in the determination of genetic diversity, ii) The genetic diversity should be increased through the collection of genetic resources iii) genic EST-SSR markers in this study should be considered in breeding programmes by using them in QTL studies.