BACKGROUND: Upland cotton is one of the utmost significant strategic fiber crops, and play a vital role in the global textile industry. METHODS AND RESULTS: A total of 128 genotypes comprised Gossypium hirsutum L, Gossypium barbadense L., and pure lines were used to examine genetic diversity using iPBS-retrotransposon markers system. Eleven highly polymorphic primers yielded 287 bands and 99.65% polymorphism was recorded. The mean polymorphism information content was estimated at 0.297 and the average diversity indices for the effective number of alleles, Shannon's information index, and overall gene diversity were 1.481, 0.443, and 0.265, respectively. The analysis of molecular variance (AMOVA) revealed that 69% of the genetic variation was within the population. A model-based STRUCTURE algorithm divided the entire germplasm into four populations and one un-classified population, the genotypes G42 (originating in Egypt) and G128 (originating in the United States), showed the highest genetic distance (0.996) so these genotypes could be suggested for breeding programs as parental lines. CONCLUSIONS: This is the first investigation using an iPBS-retrotransposon marker system to examine the genetic diversity and population structure of upland cotton germplasm. The rich diversity found in upland cotton germplasm could be exploited as a genetic resource when developing breeding programs and could also help with efforts to breed cotton around the world. These findings also show the applicability and effectiveness of iPBS-retrotransposons for the molecular characterization of cotton germplasm.